Protoxin -ii variants and methods of use

ABSTRACT

The present invention relates to Protoxin-II variants, polynucleotides encoding them, and methods of making and using the foregoing.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/886,100 filed 3 Oct. 2013, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to Protoxin-II variants, syntheticpolynucleotides encoding them, and methods of making and using theforegoing.

BACKGROUND OF THE INVENTION

Voltage-gated sodium channels (VGSC) are present in all excitable cellsincluding cardiac and skeletal muscle cells and central and peripheralneurons. In neuronal cells, sodium channels are responsible foramplifying sub-threshold depolarizations and generating the rapidupstroke of the action potential. As such, sodium channels are essentialto the initiation and propagation of electrical signals in the nervoussystem. Aberrant sodium channel function is thought to underlie avariety of medical disorders (Hubner and Jentsch, Hum Mol Genet11:2435-45, 2002), including epilepsy (Yogeeswari et al., Curr DrugTargets 5:589-602, 2004), arrhythmia (Tfelt-Hansen et al., J CardiovascElectrophysiol 21:107-15, 2010), myotonia (Cannon and Bean, J ClinInvest 120:80-3, 2010), and pain (Cregg et al., J Physiol 588:1897-904,2010). Sodium channels are typically a complex of various subunits, theprinciple one being the pore-forming alpha-subunit, which is alonesufficient for function.

Nine known members of the family of voltage-gated sodium channel alphasubunits exist in humans, Nav1.1-Nav1.9. The Nav1.x subfamily can bepharmacologically subdivided into two groups, the tetrodotoxin(TTX)-sensitive and TTX-resistant. Nav1.7, (a.k.a. PN1 or hNE) isencoded by the SCN9A gene, is TTX-sensitive and is primarily expressedin peripheral sympathetic and sensory neurons. Nav1.7 accumulates atnerve fiber endings and amplifies small sub-threshold depolarizationsand acts as a threshold channel that regulates excitability.

Nav1.7 function is implicated in various pain states, including acute,inflammatory and/or neuropathic pain. In man, gain of function mutationsof Nav1.7 have been linked to primary erythermalgia (PE), a diseasecharacterized by burning pain and inflammation of the extremities (Yanget al., J Med Genet 41:171-4, 2004), and paroxysmal extreme paindisorder (PEPD) (Fertleman et al., Neuron 52:767-74, 2006). Consistentwith this observation, non-selective sodium channel blockers lidocaine,mexiletine and carbamazepine can provide symptomatic relief in thesepainful disorders (Legroux-Crespel et al., Ann Dermatol Venereol130:429-33, 2003; Fertleman et al., Neuron 52:767-74, 2006).

Loss-of-function mutations of Nav1.7 in humans cause congenitalindifference to pain (CIP), a rare autosomal recessive disordercharacterized by a complete indifference or insensitivity to painfulstimuli (Cox et al., Nature 444:894-8, 2006; Goldberg et al, Clin Genet71:311-9, 2007; Ahmad et al., Hum Mol Genet 16:2114-21, 2007).

Single nucleotide polymorphisms in the coding region of SCN9A have beenassociated with increased nociceptor excitability and pain sensitivity.For example, a polymorphism rs6746030 resulting in R1150W substitutionin human Nav1.7 has been associated with osteoarthritis pain, lumbardiscectomy pain, phantom pain, and pancreatitis pain (Reimann et al.,Proc Natl Acad Sci USA 107:5148-53, 2010). DRG neurons expressing theR1150W mutant Nav1.7 display increased firing frequency in response todepolarization (Estacion et al., Ann Neurol 66:862-6, 2009). A disablingform of fibromyalgia has been associated with SCN9A sodium channelpolymorphism rs6754031, indicating that some patients with severefibromyalgia may have a dorsal root ganglia sodium channelopathy(Vargas-Alarcon et al., BMC Musculoskelet Disord 13:23, 2012).

In mice, deletion of the SCN9A gene in nociceptive neurons leads toreduction in mechanical and thermal pain thresholds and reduction orabolition of inflammatory pain responses (Nassar et al., Proc Natl AcadSci USA 101:12706-11, 2004). Ablating SCN9A in all sensory neuronsabolished mechanical pain, inflammatory pain and reflex withdrawalresponses to heat. Deleting SCN9A in both sensory and sympatheticneurons abolished mechanical, thermal and neuropathic pain, andrecapitulated the pain-free phenotype seen in humans with Nav1.7loss-of-function mutations (Minett et al., Nat Commun 3:791, 2012).Nav1.7 inhibitors or blockers may therefore be useful in the treatmentof a wide range of pain associated with various disorders.

Spider venoms are known to contain a large number of sodium channelblocking peptides, including Huwentoxin-IV (HwTx-IV) (Peng et al., JBiol Chem 277:47564-71, 2002), Protoxin-I, Protoxin-II (Middleton etal., Biochemistry 41:14734-47, 2002) and Phrixotoxin-III (Bosmans etal., Mol Pharmacol 69:419-29, 2006). There is a need for identificationof additional Nav1.7 blockers for treatment of a wide range of painindications. In particular, there is a need for new Nav1.7 blockers withselectivity for Nav1.7 over other voltage gated sodium channel isoforms.

SUMMARY OF THE INVENTION

One embodiment of the invention is an isolated Protoxin-II variantcomprising the sequence

-   -   X₁X₂X₃CX₄X₅WX₆QX₇CX₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄FX₁₅CX₁₆LWCX₁₇KKLW (SEQ        ID NO: 403), wherein        -   X₁ is G, P, A or deleted;        -   X₂ is P, A or deleted;        -   X₃ is S, Q, A, R or Y;        -   X₄ is Q, R, K, A or S;        -   X₅ is K, S, Q or R;        -   X₆ is M or F;        -   X₇ is T, S, R, K or Q;        -   X₈ is D or T;        -   X₉ is S, A or R;        -   X₁₀ is E, R, N, K, T or Q;        -   X₁₁ is R or K;        -   X₁₂ is K, Q, S or A;        -   X₁₃ is E, Q or D;        -   X₁₄ is G or Q;        -   X₁₅ is V or S;        -   X₁₆ is R or T; and        -   X₁₇ is K or R;        -   optionally having an N-terminal extension or a C-terminal            extension,        -   wherein the polypeptide inhibits human Nav1.7 activity with            an IC₅₀ value of about 1×10⁻⁷ M or less,        -   wherein the IC₅₀ value is measured using a FLIPR® Tetra            membrane depolarization assay using fluorescence resonance            energy transfer (FRET) in the presence of 25×10⁻⁶ M            3-veratroylveracevine in HEK293 cells stably expressing            human Nav1.7.        -   Another embodiment of the invention is an isolated            Protoxin-II variant comprising the amino acid sequence that            is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%            identical to the amino acid sequence of SEQ ID NO: 78            (GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH), wherein    -   the amino acid sequence has Q at position 1, Q at position 7 and        F at position 19, when residue numbering is according to SEQ ID        NO: 1;    -   the polypeptide inhibits human Nav1.7 activity with an IC₅₀        value of about 30×10⁻⁹ M or less, wherein the IC₅₀ value is        measured using a FLIPR® Tetra membrane depolarization assay        using fluorescence resonance energy transfer (FRET) in the        presence of 25×10⁻⁶ M 3-veratroylveracevine in HEK293 cells        stably expressing human Nav1.7; and    -   the polypeptide selectively inhibits Nav1.7.

Another embodiment of the invention is a polynucleotide encoding theProtoxin-II variant of the invention.

Another embodiment of the invention is a vector comprising the isolatedpolynucleotide of the invention.

Another embodiment of the invention is a host cell comprising the vectorof the invention.

Another embodiment of the invention is a method for producing theProtoxin-II variant of the invention, comprising culturing the host cellof the invention and recovering the Protoxin-II variant from the cell.

Another embodiment of the invention is a pharmaceutical compositioncomprising the isolated Protoxin-II variant of the invention and apharmaceutically acceptable excipient.

Another embodiment of the invention is a method of treatingNav1.7-mediated pain in a subject, comprising administering to a subjectan effective amount of the Protoxin-II variant of the invention to treatthe Nav1.7-mediated pain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the genus amino acid sequence of Protoxin-II variants thatinhibit Nav1.7 with an IC₅₀ value of 30 nM or less in a FLIPR Tetraassay. Residue numbering is according to wild-type Protoxin-II of SEQ IDNO: 1. Genus SEQ ID NO: 403.

FIG. 2 shows the IC₅₀ values for Nav1.7 and Nav1.6 inhibition in aQPatch assay, and selectivity of each variant calculated by ratio ofIC₅₀ (Nav1.6)/IC₅₀(Nav1.7) obtained in QPatch assay. SE: standard error.

FIG. 3 shows the sequences and the genus sequence of Protoxin-IIvariants that inhibit Nav1.7 with an IC₅₀ value of 30 nM or less in aFLIPR Tetra assay, and are over 30-fold selective over Nav1.6.Selectivity of each variant was calculated by ratio of IC₅₀(Nav1.6)/IC₅₀9 av1.7) obtained in QPatch assay. Residue numbering isaccording to wild-type Protoxin-II of SEQ ID NO: 1.

FIG. 4A shows efficacy of NV1D3034 (NV1D3034-OH) (SEQ ID NO: 78) againstCFA-induced thermal hyperalgesia assessed by measurement of pawwithdrawal latency in the Hargreaves test before (pre-CFA) and after CFAinjection (0) and 1-day after peptide administration (1). ***P<0.001 vs.PBS, two-way ANOVA followed by Bonferroni's multiple comparison.

FIG. 4B shows efficacy of NV1D3034 (NV1D3034-OH) (SEQ ID NO: 78) inCFA-induced thermal hyperalgesia expressed as percent MPE (maximumpossible effect) (MPE %) at each dose on day1 following peptideadministration. *P<0.05 vs PBS, one-way ANOVA followed by Bonferroni'smultiple comparison.

FIG. 5A shows efficacy of NV1D3368 (NV1D3368-OH) (SEQ ID NO: 198)against CFA-induced thermal hyperalgesia assessed by measurement of pawwithdrawal latency in the Hargreaves test before (pre-CFA) and after CFAinjection (0) and 1-day after peptide administration (1). **P<0.01 and****P<0.0001 vs. PBS, two-way ANOVA followed by Bonferroni's multiplecomparison

FIG. 5B shows efficacy of NV1D3368 (NV1D3368-OH) (SEQ ID NO: 198) inCFA-induced thermal hyperalgesia expressed as percent MPE (MPE %) ateach dose on day1 following peptide administration. *P<0.05 and **P<0.01vs PBS, one-way ANOVA followed by Bonferroni's multiple comparison.

FIG. 6A shows efficacy of NV1D2775-OH (SEQ ID NO: 56) againstCFA-induced thermal hyperalgesia assessed by measurement of pawwithdrawal latency in the Hargreaves test before (pre-CFA) and after CFAinjection (0) and 1-day after peptide administration (1). ****P<0.0001vs. PBS, two-way ANOVA followed by Bonferroni's multiple comparison.

FIG. 6B shows efficacy of NV1D2775-OH (SEQ ID NO: 56) in CFA-inducedthermal hyperalgesia expressed as percent MPE (MPE %) at each dose onday1 following peptide administration. ***P<0.001 and ****P<0.0001 vsPBS, one-way ANOVA followed by Bonferroni's multiple comparison.

FIG. 6C shows efficacy of NV1D2775-OH (SEQ ID NO: 56) againstCFA-induced tactile allodynia. Tactile thresholds of hind paw before(pre-CFA) and after CFA (0) and 1-day after peptide administration (1).****P<0.0001 vs. PBS, two-way ANOVA followed by Bonferroni's multiplecomparison.

FIG. 6D shows efficacy of NV1D2775-OH (SEQ ID NO: 56) againstCFA-induced tactile allodynia expressed as percent MPE (MPE %) on day1following peptide. ***P<0.001 vs PBS, one-way ANOVA followed byBonferroni's multiple comparison.

FIG. 7A shows time course of NV1D2775-OH mediated reversal of thermalhyperalgesia in the CFA model as assessed by measurement of pawwithdrawal latency in the Hargreaves test before and after CFA and atvarious time points post-peptide administration. **P<0.01 vs. PBS,two-way ANOVA followed by Bonferroni's multiple comparison. Shaded areasindicate compound delivery period (0-24 hr).

FIG. 7B shows time course of NV1D2775-OH mediated reversal of tactileallodynia in the CFA model as assessed by measurement of tactilethreshold before and after CFA and at various time points post-peptideadministration. **P<0.01 vs. PBS, two-way ANOVA followed by Bonferroni'smultiple comparison. Shaded areas indicate compound delivery period(0-24 hr).

FIG. 8 shows that NV1D2775-OH produced significant analgesia in thehotplate test. Thermal withdrawal latency was evaluated at 50 and 55° C.pre- and post-pump implantation. Pump implantation had no impact on thelatency in the control PBS group. One day after pump, NV1D2775-OHtreated-mice exhibited prolonged latency compared to the PBS group.*P<0.05 and ****P<0.0001 vs. PBS, one-way ANOVA followed by Bonferroni'smultiple comparison.

FIG. 9 shows that NV1D2775-OH pretreatment protected animals fromcarrageenan-induced thermal hyperalgesia. Paw withdrawal latencies weremeasured pre- and on day1 post-pump before intraplantar carrageenaninjection. Latencies were measured again at 2, 3 and 4 hr followingcarrageenan.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth.

As used herein and in the claims, the singular forms “a,” “and,” and“the” include plural reference unless the context clearly dictatesotherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which an invention belongs. Although any compositions andmethods similar or equivalent to those described herein can be used inthe practice or testing of the invention, exemplary compositions andmethods are described herein.

The term “polypeptide” means a molecule that comprises at least twoamino acid residues linked by a peptide bond to form a polypeptide.Small polypeptides of less than 50 amino acids may be referred to as“peptides”. Polypeptides may also be referred as “proteins”.

The term “polynucleotide” means a molecule comprising a chain ofnucleotides covalently linked by a sugar-phosphate backbone or otherequivalent covalent chemistry. Double and single-stranded DNAs and RNAsare typical examples of polynucleotides.

The term “complementary sequence” means a second isolated polynucleotidesequence that is antiparallel to a first isolated polynucleotidesequence and that comprises nucleotides complementary to the nucleotidesin the first polynucleotide sequence.

The term “vector” means a non-natural polynucleotide capable of beingduplicated within a biological system or that can be moved between suchsystems. Vector polynucleotides typically contain a cDNA encoding aprotein of interest and additional elements, such as origins ofreplication, polyadenylation signal or selection markers, that functionto facilitate the duplication or maintenance of these polynucleotides ina biological system. Examples of such biological systems may include acell, virus, animal, plant, and reconstituted biological systemsutilizing biological components capable of duplicating a vector. Thepolynucleotide comprising a vector may be DNA or RNA molecules or ahybrid of these.

The term “expression vector” means a vector that can be utilized in abiological system or a reconstituted biological system to direct thetranslation of a polypeptide encoded by a polynucleotide sequencepresent in the expression vector.

The term “variant” as used herein refers to a polypeptide or apolynucleotide that differs from wild type Protoxin-II polypeptide ofSEQ ID NO: 1 or the polynucleotide encoding the wild type Protoxin-IIhaving the sequence of SEQ ID NO: 107 by one or more modifications forexample, substitutions, insertions or deletions of nucleotides or aminoacids.

Throughout the specification, residues that are substituted in theProtoxin-II variants are numbered corresponding to their position in thewild-type Protoxin-II of SEQ ID NO: 1. For example, “Y1A” in thespecification refers to the substitution of tyrosine at residue positionthat corresponds to the position 1 in the wild type Protoxin-II of SEQID NO:1 with alanine.

“Complementary DNA” or “cDNA” refers to a well-known syntheticpolynucleotide that shares the arrangement of sequence elements found innative mature mRNA species with contiguous exons, with the interveningintrons present in genomic DNA are removed. The codons encoding theinitiator methionine may or may not be present in cDNA. cDNA may besynthesized for example by reverse transcription or synthetic geneassembly.

“Synthetic” or “non-natural” as used herein refers to a polynucleotideor a polypeptide molecule not present in nature.

“Nav1.7” (also referred to as hNE or PN1) or “hNav1.7” as used hereinrefers to the well-known human sodium channel protein type 9 subunitalpha having a sequence shown in GenBank accession number NP 002968.1and in SEQ ID NO: 79.

The term “wild type Protoxin-II” or “wild type ProTx-II” as used hereinrefers to the tarantula Thrixopelma pruriens (Peruvian green velvettarantula) toxin peptide having the amino acid sequenceYCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH (SEQ ID NO: 1) as described inMiddleton et al., Biochemistry 41(50):14734-47, 2002.

The term “recombinant Protoxin-II” or recombinant ProTx-II″ as usedherein refers to the recombinant Protoxin-II obtained from expressionand subsequent cleavage of a Protoxin-II fusion protein having thesequence of GPYCQKWMWTCDSERKCCEGMVCRLWCKKKLW-OH as shown in SEQ ID NO:2. Recombinant Protoxin-II incorporates a two amino acid N-terminalextension (residues G and P) when compared to the wild type Protoxin-II.

“Blocks human Nav1.7 activity” or “inhibits human Nav1.7 activity” asused herein refers to the ability of the Protoxin-II variant of theinvention to reduce membrane depolarization induced by veratridine(3-veratroylveracevine) with an IC₅₀ value of about 1×10⁻⁷ M or less ina FLIPR® Tetra membrane depolarization assay using fluorescenceresonance energy transfer (FRET), where veratridine-induceddepolarization is measured as a reduction in FRET signal usingDISBAC2(3) ([bis-(1,3-diethylthiobarbituric acid) trimethine oxonol]) asan acceptor and PTS18 (trisodium8-octadecyloxypyrene-1,3,6-trisulfonate) as a donor by exciting thedonor at 390-420 nm and measuring FRET at 515-575 nm in a cell linestably expressing human Nav1.7.

“FLIPR® Tetra membrane depolarization assay” as used herein is the assaydescribed in Example 3.

The term “substantially identical” as used herein means that the twoProtoxin-II variant amino acid sequences being compared are identical orhave “insubstantial differences”. Insubstantial differences aresubstitutions of 1, 2, 3, 4, 5, 6, or 7 amino acids in the Protoxin-IIvariant amino acid sequence that do not adversely affect peptideproperties. Amino acid sequences substantially identical to theProtoxin-II variants disclosed herein are within the scope of theapplication. In some embodiments, the sequence identity can be about80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or higher. Percent identity can bedetermined for example by pairwise alignment using the default settingsof the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carslbad,Calif.). The protein sequences of the present invention may be used as aquery sequence to perform a search against public or patent databases,for example, to identify related sequences. Exemplary programs used toperform such searches are the XBLAST or BLASTP programs(http_//www_ncbi_nlm/nih_gov), or the GenomeQuest™ (GenomeQuest,Westborough, Mass.) suite using the default settings.

Conventional one and three-letter amino acid codes are used herein asshown in Table 1.

TABLE 1 Amino acid Three letter code One letter code Alanine Ala AArginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys C GlutamateGlu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V

The present invention provides isolated Protoxin-II (ProTx-II) variantpolypeptides that inhibit human Nav1.7 activity, polynucleotidesencoding them, vectors, host cells, and methods of using thepolynucleotides and polypeptides of the invention. The polypeptides ofthe invention inhibit depolarization resulting from Nav1.7 activation,and therefore may be useful in the treatment of various conditionsassociated with pain and conditions associated with sensory orsympathetic neuron dysfunction.

The variants of the invention are potent inhibitors of Nav1.7. Thecurrent invention is based, at least in part, on the finding thatcertain residue substitutions in Protoxin-II enhance selectivity,synthetic yield and/or homogeneity without adversely affecting thepotency of the generated Protoxin-II variants, specifically W7 and M19,and additionally residues Y1 and S11, and further additionally residuesE12, R22 and (residue numbering according to SEQ ID NO: 1).

One embodiment of the invention is an isolated Protoxin-II variant,wherein the Protoxin-II variant inhibits human Nav1.7 activity with anIC₅₀ value of about 1×10⁻⁷ M or less, about 1×10⁻⁹ M or less, about1×10⁻⁹ M or less, about 1×10⁻¹⁰ M or less, about 1×10⁻¹¹ M or less, orabout 1×10⁻¹² M or less, wherein the IC₅₀ value is measured using aFLIPR® Tetra membrane depolarization assay using fluorescence resonanceenergy transfer (FRET) in the presence of 25×10⁻⁶ M3-veratroylveracevine in HEK293 cells stably expressing human Nav1.7.

Another embodiment of the invention is an isolated Protoxin-II variantcomprising the sequence

-   -   X₁X₂X₃CX₄X₅WX₆QX₇CX₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄FX₁₅CX₁₆LWCX₁₇KKLW (SEQ        ID NO: 403), wherein        -   X₁ is G, P, A or deleted;        -   X₂ is P, A or deleted;        -   X₃ is S, Q, A, R or Y;        -   X₄ is Q, R, K, A or S;        -   X₅ is K, S, Q or R;        -   X₆ is M or F;        -   X₇ is T, S, R, K or Q;        -   X₈ is D or T;        -   X₉ is S, A or R;        -   X₁₀ is E, R, N, K, T or Q;        -   X₁₁ is R or K;        -   X₁₂ is K, Q, S or A;        -   X₁₃ is E, Q or D;        -   X₁₄ is G or Q;        -   X₁₅ is V or S;        -   X₁₆ is R or T; and        -   X₁₇ is K or R;        -   optionally having an N-terminal extension or a C-terminal            extension,        -   wherein the polypeptide inhibits human Nav1.7 activity with            an IC₅₀ value of about 1×10⁻⁷ M or less,        -   wherein the IC₅₀ value is measured using a FLIPR® Tetra            membrane depolarization assay using fluorescence resonance            energy transfer (FRET) in the presence of 25×10⁻⁶ M            3-veratroylveracevine in HEK293 cells stably expressing            human Nav1.7.

In some embodiments, the N-terminal extension comprises the amino acidsequences of SEQ ID NOs: 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384 or 385.

In some embodiments, the C-terminal extension comprises the amino acidsequence of SEQ ID NOs: 374, 386, 387, 388, 389, 390, 391, 392, 393,394, 395, 396 or 397.

In some embodiments, the N-terminal and/or the C-terminal extension isconjugated to the Protoxin-II variant via a linker.

In some embodiments, the linker comprises the amino acid sequence of SEQID NOs: 383, 392, 398, 399, 400, 401 or 402.

In some embodiments, the N-terminal extension consists of the amino acidsequences of SEQ ID NOs: 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384 or 385.

In some embodiments, the C-terminal extension consists of the amino acidsequence of SEQ ID NOs: 374, 386, 387, 388, 389, 390, 391, 392, 393,394, 395, 396 or 397.

In some embodiments, the linker consists of the amino acid sequence ofSEQ ID NOs: 383, 392, 398, 399, 400, 401 or 402.

The Protoxin-II variants of the invention are potent Nav1.7 inhibitors.Recombinant Protoxin-II (SEQ ID NO: 2) has an IC₅₀ value of about 4×10⁻⁹M for human Nav1.7 in a veratridine-induced depolarization inhibitionassay measuring decline in FRET (fluorescence resonance energy transfer)in cells stably expressing Nav1.7 using FLIPR® Tetra instrument(Molecular Devices) using experimental details described in Example 3. AProtoxin-II variant is “a potent” Nav1.7 inhibitor when the IC₅₀ valuein the assay described above and in Experiment 3 is about 30×10⁻⁹ M orless i.e. within 10 fold of recombinant Protoxin-II. For clarity, anIC₅₀ of 30×10⁻⁹ M is identical to IC₅₀ of 3.0×10⁻⁸ M.

The Protoxin-II variant polypeptides of the invention may be produced bychemical synthesis, such as solid phase peptide synthesis, on anautomated peptide synthesizer. Alternatively, the polypeptides of theinvention may be obtained from polynucleotides encoding the polypeptidesby the use of cell-free expression systems such as reticulocyte lysatebased expression systems, or by recombinant expression systems. Thoseskilled in the art will recognize other techniques for obtaining thepolypeptides of the invention. In an exemplary method, the Protoxin-IIvariants of the invention are generated by expressing them as humanserum albumin (HSA) fusion proteins utilizing a glycine-rich linker suchas (GGGGS)₄ (SEQ ID NO:80) or (GGGGS)₆ (SEQ ID NO: 81) coupled to aprotease cleavable linker such as a recognition sequence for HRV3Cprotease (Recombinant type 14 3C protease from human rhinovirus)LEVLFQGP (HRV3C linker) (SEQ ID NO: 82), and cleaving the expressedfusion proteins with the HRV3C protease to release the recombinantProtoxin-II variant peptides. Hexahistidine (SEQ ID NO: 108) or othertags may be used to facilitate purification using well known methods.

Protoxin-II variants of the invention may be purified using methodsdescribed herein. In an exemplary method, Protoxin-II variants of theinvention expressed as HSA fusion proteins and cleaved with HRV3Cprotease may be purified using sold phase extraction (SPE) as describedherein.

Generation of the Protoxin-II variants optionally having N-terminaland/or C-terminal extensions, and Protoxin-II variant fusion proteins istypically achieved at the nucleic acid level. The polynucleotides may besynthesized using chemical gene synthesis according to methods describedin U.S. Pat. Nos. 6,521,427 and 6,670,127, utilizing degenerateoligonucleotides to generate the desired variants, or by standard PCRcloning and mutagenesis. Libraries of variants may be generated bystandard cloning techniques to clone the polynucleotides encoding theProtoxin-II variants into the vector for expression.

The Protoxin-II variants may incorporate additional N- and/or C-terminalamino acids when compared to the wild type Protoxin-II of SEQ ID NO: 1,for example resulting from cloning and/or expression schemes. Forexample, cleavage from HSA after expression of the variant asHSA-linker-HRV3C cleavable peptide-Protoxin-II variant fusion proteinmay result in the incorporation of additional two residues to theN-terminus of each Protoxin-II variant, such as G and P.

The Protoxin-II variants of the invention are tested for their abilityto inhibit human Nav1.7 using methods described herein. An exemplaryassay is a veratridine-induced depolarization inhibition assay measuringdecline in FRET (fluorescence resonance energy transfer) in cells stablyexpressing Nav1.7. Another exemplary assay employs electrophysiologicalrecordings to measure changes in Nav1.7-mediated currents using wellknown patch clamp techniques and as described herein.

Another embodiment of the invention is an isolated Protoxin-II variantcomprising the amino acid sequence of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 35, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368 367, 370 or 371.

The Protoxin-II variants of the invention may inhibit human Nav1.7 withan IC₅₀ value of about 1×10⁻⁷ M or less, about 1×10⁻⁸ M about 1×10⁻⁹ orless, about 1×10⁻¹° M or less, about 1×10⁻¹¹ M or less, or about 1×10⁻¹²M or less. Exemplary variants demonstrating the range of IC₅₀ values arevariants having amino acid sequences shown in SEQ ID NOs: 30, 40, 44,52, 56, 56, 59, 65, 78, 109, 110, 111, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 162, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 177, 178, 179, 180, 182, 183, 184, 185,186, 189, 190, 193, 195, 197, 199, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 224, 226, 227, 231, 232, 243, 244, 245,247, 249, 252, 255, 258, 261, 263, 264, 265, 266, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 332, 334,335, 336, 337, 339, 340, 341, 342, 346, 351, 358, 359, 364, 366, 367, or368.

Table 2 and Table 3 show the sequences of select Protoxin-II variants.

TABLE 2  Protoxin-II variant SEQ Protein peptide ID name name NO:Protein amino acid sequence wild type 1YCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH NV1D12 2GPYCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH NV1D748 3GPACQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH NV1D751 4GPQCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH NV1D2292 5GPRCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH NV1D750 6GPSCQKWMWTCDSERKCCEGMVCRLWCKKKLW-COOH NV1D1328 7GPYCQKWFWTCDSERKCCEGMVCRLWCKKKLW-COOH NV1D774 8GPYCQKWMQTCDSERKCCEGMVCRLWCKKKLW-COOH NV1D786 9GPYCQKWMWTCDAERKCCEGMVCRLWCKKKLW-COOH NV1D2300 10GPYCQKWMWTCDRERKCCEGMVCRLWCKKKLW-COOH NV1D791 11GPYCQKWMWTCDSKRKCCEGMVCRLWCKKKLW-COOH NV1D1332 12GPYCQKWMWTCDSNRKCCEGMVCRLWCKKKLW-COOH NV1D2512 13GPYCQKWMWTCDSERKCCEGFVCRLWCKKKLW-COOH NV1D1336 14GPYCQKWMWTCDSERKCCEGLVCRLWCKKKLW-COOH NV1D1337 15GPYCQKWMWTCDSERKCCEGMVCTLWCKKKLW-COOH NV1D2308 16GPYCQKWMWTCDSERKCCEGMVCRLWCRKKLW-COOH NV1G953 NV1D2670 17GPACQKWMQTCDSERKCCEGMVCRLWCKKKLW-COOH NV1G951 NV1D2674 18GPACQKWMWTCDAERKCCEGMVCRLWCKKKLW-COOH NV1G909 NV1D2664 19GPACQKWMWTCDSERKCCEGFVCRLWCKKKLW-COOH NV1G963 NV1D2671 20GPQCQKWMQTCDSERKCCEGMVCRLWCKKKLW-COOH NV1G949 NV1D2675 21GPQCQKWMWTCDAERKCCEGMVCRLWCKKKLW-COOH NV1G977 NV1D2665 22GPQCQKWMWTCDSERKCCEGFVCRLWCKKKLW-COOH NV1G957 NV1D2668 23GPRCQKWMQTCDSERKCCEGMVCRLWCKKKLW-COOH NV1G965 NV1D2672 24GPRCQKWMWTCDAERKCCEGMVCRLWCKKKLW-COOH NV1G973 NV1D2662 25GPRCQKWMWTCDSERKCCEGFVCRLWCKKKLW-COOH NV1G975 NV1D2669 26GPSCQKWMQTCDSERKCCEGMVCRLWCKKKLW-COOH NV1G971 NV1D2673 27GPSCQKWMWTCDAERKCCEGMVCRLWCKKKLW-COOH NV1G995 NV1D2663 28GPSCQKWMWTCDSERKCCEGFVCRLWCKKKLW-COOH NV1G961 NV1D2676 29GPYCQKWMQTCDAERKCCEGMVCRLWCKKKLW-COOH NV1G911 NV1D2666 30GPYCQKWMQTCDSERKCCEGFVCRLWCKKKLW-COOH NV1D2816 31GPACQKWFQTCDSERKCCEGMVCRLWCKKKLW-COOH NV1G905 NV1D2735 32GPACQKWMQTCDSERKCCEGFVCRLWCKKKLW-COOH NV1G919 NV1D2739 33GPACQKWMWTCDAERKCCEGFVCRLWCKKKLW-COOH NV1G979 NV1D2731 34GPACQKWMQTCDAERKCCEGMVCRLWCKKKLW-COOH NV1D2810 35GPQCQKWFQTCDSERKCCEGMVCRLWCKKKLW-COOH NV1G1099 NV1D2732 36GPQCQKWMQTCDAERKCCEGMVCRLWCKKKLW-COOH NV1G1011 NV1D2740 37GPQCQKWMWTCDAERKCCEGFVCRLWCKKKLW-COOH NV1D2819 38GPRCQKWFWTCDAERKCCEGMVCRLWCKKKLW-COOH NV1G1105 NV1D2729 39GPRCQKWMQTCDAERKCCEGMVCRLWCKKKLW-COOH NV1G1013 NV1D2733 40GPRCQKWMQTCDSERKCCEGFVCRLWCKKKLW-COOH NV1D2814 41GPSCQKWFQTCDSERKCCEGMVCRLWCKKKLW-COOH NV1D2820 42GPSCQKWFWTCDAERKCCEGMVCRLWCKKKLW-COOH NV1G983 NV1D2730 43GPSCQKWMQTCDAERKCCEGMVCRLWCKKKLW-COOH NV1G1003 NV1D2734 44GPSCQKWMQTCDSERKCCEGFVCRLWCKKKLW-COOH NV1G1009 NV1D2738 45GPSCQKWMWTCDAERKCCEGFVCRLWCKKKLW-COOH NV1D2851 46GPYCQKWFKTCDAERKCCEGMVCRLWCKKKLW-COOH NV1D2850 47GPYCQKWFQTCDAERKCCEGMVCRLWCKKKLW-COOH NV1G987 NV1D2667 48GPYCQKWMWTCDAERKCCEGFVCRLWCKKKLW-COOH NV1D2867 49GPACQKWFQTCDAERKCCEGMVCRLWCKKKLW-COOH NV1D2881 50GPACQKWFQTCDSERKCCEGFVCRLWCKKKLW-COOH NV1D2882 51GPACQKWFQTCDSERKCCEGLVCRLWCKKKLW-COOH NV1G899 NV1D2774 52GPACQKWMQTCDAERKCCEGFVCRLWCKKKLW-COOH NV1G1077 NV1D2902 53GPACQKWMQTCDAERKCCEGLVCRLWCKKKLW-COOH NV1D2861 54GPQCQKWFQTCDAERKCCEGMVCRLWCKKKLW-COOH NV1D2870 55GPQCQKWFQTCDSERKCCEGLVCRLWCKKKLW-COOH NV1G1007 NV1D2775 56GPQCQKWMQTCDAERKCCEGFVCRLWCKKKLW-COOH NV1G1067 NV1D2893 57GPQCQKWMQTCDAERKCCEGLVCRLWCKKKLW-COOH NV1D2887 58GPRCQKWFWTCDAERKCCEGFVCRLWCKKKLW-COOH NV1G1005 NV1D2772 59GPRCQKWMQTCDAERKCCEGFVCRLWCKKKLW-COOH NV1G1061 NV1D2896 60GPRCQKWMQTCDAERKCCEGLVCRLWCKKKLW-COOH NV1D2877 61GPSCQKWFQTCDSERKCCEGFVCRLWCKKKLW-COOH NV1D2878 62GPSCQKWFQTCDSERKCCEGLVCRLWCKKKLW-COOH NV1D2889 63GPSCQKWFWTCDAERKCCEGFVCRLWCKKKLW-COOH NV1D2889 64GPSCQKWFWTCDAERKCCEGFVCRLWCKKKLW-COOH NV1G1001 NV1D2773 65GPSCQKWMQTCDAERKCCEGFVCRLWCKKKLW-COOH NV1D2890 66GPSCQKWFWTCDAERKCCEGLVCRLWCKKKLW-COOH NV1G1109 NV1D2899 67GPSCQKWMQTCDAERKCCEGLVCRLWCKKKLW-COOH NV1D2905 68GPYCQKWFQTCDAERKCCEGFVCRLWCKKKLW-COOH NV1D2906 69GPYCQKWFQTCDAERKCCEGLVCRLWCKKKLW-COOH NV1D2921 70GPACQKWFQTCDAERKCCEGFVCRLWCKKKLW-COOH NV1D2922 71GPACQKWFQTCDAERKCCEGLVCRLWCKKKLW-COOH NV1D2909 72GPQCQKWFQTCDAERKCCEGFVCRLWCKKKLW-COOH NV1D2910 73GPQCQKWFQTCDAERKCCEGLVCRLWCKKKLW-COOH NV1D2913 74GPRCQKWFQTCDAERKCCEGFVCRLWCKKKLW-COOH NV1D2914 75GPRCQKWFQTCDAERKCCEGLVCRLWCKKKLW-COOH NV1D2917 76GPSCQKWFQTCDAERKCCEGFVCRLWCKKKLW-COOH NV1D2918 77GPSCQKWFQTCDAERKCCEGLVCRLWCKKKLW-COOH NV1G1153 NV1D3034 78GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH

TABLE 3  Protoxin-II variant SEQ ID Protein name peptide name NO:Protein amino acid sequence (-GP) NV1G1001 (-GP) NV1D2773 109SCQKWMQTCDAERKCCEGFVCRLW CKKKLW-COOH (-GP) NV1G1001- (-GP) NV1D2773-NH2110 SCQKWMQTCDAERKCCEGFVCRLW NH-Me CKKKLW-NH2 NV1G1007-NH2 NV1D2775-NH2111 GPQCQKWMQTCDAERKCCEGFVCR LWCKKKLW-NH2 NV1G1107-NH2 NV1D2890-NH2 112SCQKWFWTCDAERKCCEGLVCRLW CKKKLW-NH2 NV1G1137 NV1D2974 113GPQCQKWMQTCDAERKCCEGFSCT LWCKKKLW-COOH (-GP) N-Ac- (-GP) N-Ac- 114 Ac-NV1G1137-NH2  NV1D2974-NH2 QCQKWMQTCDAERKCCEGFSCTLW CKKKLW-NH2(-GP) N-Ac- (-GP) N-Ac- 115 Ac- NV1G1137- NV1D2974QCQKWMQTCDAERKCCEGFSCTLW CKKKLW-COOH NV1G1153 NV1D3034 116GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1153-NH2 NV1D3034-NH2 117GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLW-NH2 NV1G1153-NH- NV1D3034-NH- 118GPQCQKWMQTCDRERKCCEGFVCT butyl butyl LWCRKKLW-NH-butyl NV1G1153-NH-NV1D3034-NH- 119 GPQCQKWMQTCDRERKCCEGFVCT methyl methylLWCRKKLW-NH-methyl (-GP) N-Ac- (-GP) N-Ac- 120 Ac- NV1G1153 NV1D3034QCQKWMQTCDRERKCCEGFVCTLW CRKKLW-COOH (-GP) N-Ac- (-GP) N-Ac- 121 Ac-NV1G1153-NH2 NV1D3034-NH2 QCQKWMQTCDRERKCCEGFVCTLW CRKKLW-NH2 NV1G1818NV1D3368 122 GPQCQKWMQTCDRTRKCCEGFVCT LWCRKKLW-COOH NV1G1818-NH2NV1D3368-NH2 123 GPQCQKWMQTCDRTRKCCEGFVCT LWCRKKLW-NH2 NV1G1147 NV1D2969124 GPSCQKWMQTCDAERKCCEGFSCRL WCKKKLW-COOH NV1G1145 NV1D2970 125GPSCQKWMQTCDAERKCCEGFVCT LWCKKKLW-COOH NV1G1143 NV1D2971 126GPSCQKWMQTCDAERKCCEGFSCTL WCKKKLW-COOH NV1G1141 NV1D2972 127GPQCQKWMQTCDAERKCCEGFSCR LWCKKKLW-COOH NV1G1139 NV1D2973 128GPQCQKWMQTCDAERKCCEGFVCT LWCKKKLW-COOH NV1G1137 NV1D2974 129GPQCQKWMQTCDAERKCCEGFSCT LWCKKKLW-COOH NV1G1137-NH2 NV1D2974-NH2 130GPQCQKWMQTCDAERKCCEGFSCT LWCKKKLW-NH2 NV1G1517 NV1D3004 131GPQCQKWMQTCDRERKCCEGFVCR LWCKKKLW-COOH NV1G1515 NV1D3005 132GPQCQKWMQTCDANRKCCEGFVC RLWCKKKLW-COOH NV1G1519 NV1D3006 133GPQCQKWMQTCDARRKCCEGFVCR LWCKKKLW-COOH NV1G1513 NV1D3007 134GPQCQKWMQTCDAERKCCEGFVCR LWCRKKLW-COOH NV1G1523 NV1D3012 135GPQCQKWMQTCDRNRKCCEGFVC RLWCKKKLW-COOH NV1G1525 NV1D3013 136GPQCQKWMQTCDRRRKCCEGFVCR LWCKKKLW-COOH NV1G1255 NV1D3014 137GPQCQKWMQTCDRERKCCEGFVCT LWCKKKLW-COOH NV1G1187 NV1D3015 138GPQCQKWMQTCDRERKCCEGFVCR LWCRKKLW-COOH NV1G1257 NV1D3016 139GPQCQKWMQTCDANRKCCEGFVCT LWCKKKLW-COOH NV1G1221 NV1D3017 140GPQCQKWMQTCDARRKCCEGFVCT LWCKKKLW-COOH NV1G1521 NV1D3018 141GPQCQKWMQTCDANRKCCEGFVCR LWCRKKLW-COOH NV1G1531 NV1D3019 142GPQCQKWMQTCDARRKCCEGFVCR LWCRKKLW-COOH NV1G1239 NV1D302O 143GPQCQKWMQTCDAERKCCEGFVCT LWCRKKLW-COOH NV1G1583 NV1D3030 144GPQCQKWMQTCDRNRKCCEGFVCT LWCKKKLW-COOH NV1G1527 NV1D3031 145GPQCQKWMQTCDRRRKCCEGFVCT LWCKKKLW-COOH NV1G1511 NV1D3032 146GPQCQKWMQTCDRNRKCCEGFVCR LWCRKKLW-COOH NV1G1509 NV1D3033 147GPQCQKWMQTCDRRRKCCEGFVCR LWCRKKLW-COOH NV1G1231 NV1D3035 148GPQCQKWMQTCDANRKCCEGFVCT LWCRKKLW-COOH NV1G1211 NV1D3036 149GPQCQKWMQTCDARRKCCEGFVCT LWCRKKLW-COOH NV1G1267 NV1D3044 150GPQCQKWMQTCDRNRKCCEGFVCT LWCRKKLW-COOH NV1G1269 NV1D3045 151GPQCQKWMQTCDRRRKCCEGFVCT LWCRKKLW-COOH NV1G1215 NV1D3048 152GPQCQKWMQTCDAKRKCCEGFVCR LWCKKKLW-COOH NV1G1593 NV1D3050 153GPQCQKWMQTCDRKRKCCEGFVCR LWCKKKLW-COOH NV1G1263 NV1D3051 154GPQCQKWMQTCDAKRKCCEGFVCT LWCKKKLW-COOH NV1G1585 NV1D3052 155GPQCQKWMQTCDAKRKCCEGFVCR LWCRKKLW-COOH NV1G1623 NV1D3056 156GPQCQKWMQTCDRKRKCCEGFVCT LWCKKKLW-COOH NV1G1613 NV1D3057 157GPQCQKWMQTCDRKRKCCEGFVCR LWCRKKLW-COOH NV1G1259 NV1D3058 158GPQCQKWMQTCDAKRKCCEGFVCT LWCRKKLW-COOH NV1G1265 NV1D3062 159GPQCQKWMQTCDRKRKCCEGFVCT LWCRKKLW-COOH NV1G1273 NV1D3109 160GPQCQKWMWTCDARRKCCEGFVCT LWCRKKLW-COOH NV1G1225 NV1D3121 161GPQCQKWMWTCDRKRKCCEGFVC TLWCRKKLW-COOH NV1G1886 NV1D3249 162GPAAAAAQCQKWMQTCDAERKCC EGFVCRLWCKKKLW-COOH NV1G1633 NV1D3251 163GPAPAPAQCQKWMQTCDAERKCCE GFVCRLWCKKKLW-COOH NV1G1631 NV1D3252 164GPQCQKWMQTCDAERKCCEGFVCR LWCKKKLWAPAPA-COOH NV1G1885 NV1D3254 165GPQCQKWMQTCDAERKCCEGFVCR LWCKKKLWGGGGG-COOH NV1G1884 NV1D3256 166GPCCNCSSKWCRDHSRCCGRGSAPA PAPAPAPGSQCQKWMQTCDAERKC CEGFVCRLWCKKKLW-COOHNV1G1881 NV1D3257 167 GPQCQKWMQTCDAERKCCEGFVCR LWCKKKLWGSAPAPAPAPAPGSCCNCSSKWCRDHSRCC-COOH NV1G1879 NV1D3259 168 GPQCQKWMQTCDAERKCCEGFVCRLWCKKKLWGSAPAPAPAPAPAPAPA PAPAPGSCCNCSSKWCRDHSRCCGR-COOH NV1G1883NV1D3260 169 GPCCNCSSKWCRDHSRCCGRGSAPA PAPAPAPAPAPAPAPAPGSQCQKWMQTCDAERKCCEGFVCRLWCKKKLW-COOH NV1G1880 NV1D3261 170GPQCQKWMQTCDAERKCCEGFVCR LWCKKKLWGSAPAPAPAPAPAPAPAPAPAPGSCCNCSSKWCRDHSRCC-COOH NV1G1882 NV1D3262 171GPCCNCSSKWCRDHSRCCGSAPAPA PAPAPAPAPAPAPAPGSQCQKWMQTCDAERKCCEGFVCRLWCKKKLW-COOH NV1G1776 NV1D3339 172GPQCRKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1775 NV1D3340 173GPQCKKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1768 NV1D3341 174GPQCTKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1777 NV1D3342 175GPQCAKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1770 NV1D3344 176GPQCEKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1767 NV1D3345 177GPQCSKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1769 NV1D3346 178GPQCQRWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1774 NV1D3347 179GPQCQTWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1771 NV1D3348 180GPQCQAWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1778 NV1D3349 181GPQCQDWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1773 NV1D3350 182GPQCQEWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1779 NV1D3351 183GPQCQQWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1772 NV1D3352 184GPQCQSWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1868 NV1D3353 185GPQCQKWMQRCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1824 NV1D3354 186GPQCQKWMQKCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1863 NV1D3356 187GPQCQKWMQDCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1826 NV1D3357 188GPQCQKWMQECDRERKCCEGFVCT LWCRKKLW-COOH NV1G1810 NV1D3358 189GPQCQKWMQQCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1836 NV1D3359 190GPQCQKWMQSCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1834 NV1D3360 191GPQCQKWMQTCRRERKCCEGFVCT LWCRKKLW-COOH NV1G1829 NV1D3361 192GPQCQKWMQTCKRERKCCEGFVCT LWCRKKLW-COOH NV1G1820 NV1D3362 193GPQCQKWMQTCTRERKCCEGFVCT LWCRKKLW-COOH NV1G1828 NV1D3363 194GPQCQKWMQTCARERKCCEGFVCT LWCRKKLW-COOH NV1G1827 NV1D3365 195GPQCQKWMQTCQRERKCCEGFVCT LWCRKKLW-COOH NV1G1857 NV1D3366 196GPQCQKWMQTCSRERKCCEGFVCT LWCRKKLW-COOH NV1G1823 NV1D3367 197GPQCQKWMQTCDRQRKCCEGFVCT LWCRKKLW-COOH NV1G1818 NV1D3368 198GPQCQKWMQTCDRTRKCCEGFVCT LWCRKKLW-COOH NV1G1811 NV1D3369 199GPQCQKWMQTCDREKKCCEGFVCT LWCRKKLW-COOH NV1G1853 NV1D3370 200GPQCQKWMQTCDRETKCCEGFVCT LWCRKKLW-COOH NV1G1817 NV1D3371 201GPQCQKWMQTCDREAKCCEGFVCT LWCRKKLW-COOH NV1G1814 NV1D3372 202GPQCQKWMQTCDREDKCCEGFVCT LWCRKKLW-COOH NV1G1831 NV1D3374 203GPQCQKWMQTCDREQKCCEGFVCT LWCRKKLW-COOH NV1G1681 NV1D3511 252GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWSPGARAF-COOH NV1G1693 NV1D3512 253GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWAPAPAPAPAPDGPWRK M-COOH NV1G1705NV1D3513 254 GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWAPAPADGPWRKM- COOHNV1G1689 NV1D3514 255 GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWDGPWRKM-COOHNV1G1711 NV1D3515 256 GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLWAPAPAPAPAPFGQKASS- COOH NV1G1685 NV1D3516 257GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWAPAPAFGQKASS-COOH NV1G1697 NV1D3517 258GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWFGQKASS-COOH NV1G1695 NV1D3518 259GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWAPAPAPAPAPQRFVTG HFGGLYPANG-COOHNV1G1701 NV1D3519 260 GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWAPAPAQRFVTGHFGGLYPANG-COOH NV1G1691 NV1D3520 261 GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLWQRFVTGHFGGLYPANG- COOH NV1G1679 NV1D3521 262GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWAPAPAPAPAPRRRRRRR RRRR-COOH NV1G1683NV1D3523 263 GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWRRRRRRRRRRR-COOH NV1G1707NV1D3524 264 GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWAPAPAPAPAPYGRKKRRQRRR-COOH NV1G1713 NV1D3525 265 GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLWAPAPAYGRKKRRQRRR- COOH NV1G1687 NV1D3526 266GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWYGRKKRRQRRR-COOH NV1G1699 NV1D3527 267GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWAPAPAPAPAP-COOH NV1G1675 NV1D3528 268GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWAPAPA-COOH NV1G1754 NV1D3529 269GPRCQKWMQTCDAKRKCCEGFVCT LWCRKKLW-COOH NV1G1748 NV1D3530 270GPSCQKWMQTCDAKRKCCEGFVCT LWCRKKLW-COOH NV1G1747 NV1D3531 271GPYCQKWMQTCDAKRKCCEGFVCT LWCRKKLW-COOH NV1G1752 NV1D3532 272GPACQKWMQTCDAKRKCCEGFVCT LWCRKKLW-COOH NV1G1722 NV1D3533 273GPQCQKWMQTCDAKRKCCEGFSCT LWCRKKLW-COOH NV1G1744 NV1D3534 274GPRCQKWMQTCDAKRKCCEGFSCT LWCRKKLW-COOH NV1G1742 NV1D3535 275GPSCQKWMQTCDAKRKCCEGFSCTL WCRKKLW-COOH NV1G1723 NV1D3536 276GPYCQKWMQTCDAKRKCCEGFSCTL WCRKKLW-COOH NV1G1745 NV1D3537 277GPACQKWMQTCDAKRKCCEGFSCT LWCRKKLW-COOH NV1G1757 NV1D3538 278GPRCQKWMQTCDRNRKCCEGFVCT LWCRKKLW-COOH NV1G1762 NV1D3539 279GPSCQKWMQTCDRNRKCCEGFVCT LWCRKKLW-COOH NV1G1763 NV1D3540 280GPYCQKWMQTCDRNRKCCEGFVCT LWCRKKLW-COOH NV1G1728 NV1D3541 281GPACQKWMQTCDRNRKCCEGFVCT LWCRKKLW-COOH NV1G1730 NV1D3542 282GPQCQKWMQTCDRNRKCCEGFSCT LWCRKKLW-COOH NV1G1760 NV1D3543 283GPRCQKWMQTCDRNRKCCEGFSCT LWCRKKLW-COOH NV1G1727 NV1D3544 284GPSCQKWMQTCDRNRKCCEGFSCT LWCRKKLW-COOH NV1G1729 NV1D3545 285GPYCQKWMQTCDRNRKCCEGFSCT LWCRKKLW-COOH NV1G1867 NV1D3546 286GPACQKWMQTCDRNRKCCEGFSCT LWCRKKLW-COOH NV1G1759 NV1D3547 287GPRCQKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1758 NV1D3548 288GPSCQKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1766 NV1D3549 289GPYCQKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1761 NV1D3550 290GPACQKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1726 NV1D3551 291GPRCQKWMQTCDRERKCCEGFSCTL WCRKKLW-COOH NV1G1721 NV1D3552 292GPSCQKWMQTCDRERKCCEGFSCTL WCRKKLW-COOH NV1G1765 NV1D3553 293GPYCQKWMQTCDRERKCCEGFSCTL WCRKKLW-COOH NV1G1764 NV1D3554 294GPACQKWMQTCDRERKCCEGFSCT LWCRKKLW-COOH NV1G1732 NV1D3555 295GPRCQKWMQTCDAERKCCEGFSCT LWCKKKLW-COOH NV1G1862 NV1D3556 296GPYCQKWMQTCDAERKCCEGFSCTL WCKKKLW-COOH NV1G1751 NV1D3558 297GPRCQKWMQTCDANRKCCEGFSCT LWCKKKLW-COOH NV1G1866 NV1D3559 298GPSCQKWMQTCDANRKCCEGFSCT LWCKKKLW-COOH NV1G1865 NV1D3560 299GPYCQKWMQTCDANRKCCEGFSCT LWCKKKLW-COOH NV1G1716 NV1D3561 300GPACQKWMQTCDANRKCCEGFSCT LWCKKKLW-COOH NV1G1724 NV1D3562 301GPRCQKWMQTCDARRKCCEGFSCT LWCKKKLW-COOH NV1G1717 NV1D3563 302GPSCQKWMQTCDARRKCCEGFSCTL WCKKKLW-COOH NV1G1743 NV1D3564 303GPYCQKWMQTCDARRKCCEGFSCT LWCKKKLW-COOH NV1G1720 NV1D3565 304GPACQKWMQTCDARRKCCEGFSCT LWCKKKLW-COOH NV1G1735 NV1D3566 305GPRCQKWMQTCDAERKCCEGFVCT LWCKKKLW-COOH NV1G1734 NV1D3568 306GPACQKWMQTCDAERKCCEGFVCT LWCKKKLW-COOH NV1G1741 NV1D3569 307GPRCQKWMQTCDARRKCCEGFVCT LWCKKKLW-COOH NV1G1719 NV1D3570 308GPSCQKWMQTCDARRKCCEGFVCT LWCKKKLW-COOH NV1G1718 NV1D3571 309GPYCQKWMQTCDARRKCCEGFVCT LWCKKKLW-COOH NV1G1725 NV1D3572 310GPACQKWMQTCDARRKCCEGFVCT LWCKKKLW-COOH NV1G1869 NV1D3573 311GPRCQKWMQTCDANRKCCEGFVCT LWCKKKLW-COOH NV1G1755 NV1D3574 312GPSCQKWMQTCDANRKCCEGFVCT LWCKKKLW-COOH NV1G1756 NV1D3575 313GPYCQKWMQTCDANRKCCEGFVCT LWCKKKLW-COOH NV1G1746 NV1D3576 314GPACQKWMQTCDANRKCCEGFVCT LWCKKKLW-COOH NV1G1733 NV1D3577 315GPRCQKWMQTCDAERKCCEGFSCR LWCKKKLW-COOH NV1G1738 NV1D3578 316GPYCQKWMQTCDAERKCCEGFSCR LWCKKKLW-COOH NV1G1737 NV1D3579 317GPACQKWMQTCDAERKCCEGFSCR LWCKKKLW-COOH NV1G1740 NV1D3580 318GPRCQKWMQTCDARRKCCEGFSCR LWCKKKLW-COOH NV1G1864 NV1D3581 319GPSCQKWMQTCDARRKCCEGFSCR LWCKKKLW-COOH NV1G1739 NV1D3582 320GPYCQKWMQTCDARRKCCEGFSCR LWCKKKLW-COOH NV1G1870 NV1D3583 321GPACQKWMQTCDARRKCCEGFSCR LWCKKKLW-COOH NV1G1715 NV1D3584 322GPRCQKWMQTCDANRKCCEGFSCR LWCKKKLW-COOH NV1G1753 NV1D3585 323GPSCQKWMQTCDANRKCCEGFSCR LWCKKKLW-COOH NV1G1750 NV1D3586 324GPYCQKWMQTCDANRKCCEGFSCR LWCKKKLW-COOH NV1G1750-NH2 NV1D3586-NH2 325GPYCQKWMQTCDANRKCCEGFSCR LWCKKKLW-NH2 NV1G1749 NV1D3587 326GPACQKWMQTCDANRKCCEGFSCR LWCKKKLW-COOH NV1G1871 NV1D3772 327GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWSHSNTQTLAKAPEHTG- COOH NV1G1839NV1D3774 328 GPSHSNTQTLAKAPEHTGAPAPAPA PAPAPAPAPAPAPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH NV1G1877 NV1D3775 329 GPSHSNTQTLAKAPEHTGAPAPAPAPAPQCQKWMQTCDRERKCCEGFVC TLWCRKKLW-COOH NV1G1872 NV1D3777 330GPSHSNTQTLAKAPEHTGQCQKWM QTCDRERKCCEGFVCTLWCRKKLW- COOH NV1G1941NV1D3782 331 GPQCQKWMQTCDRERKCCEGFVCT LWCRKKAW-COOH NV1G1990 NV1D3788332 GPAAAAAQCQKWMQTCDRERKCC EGFVCTLWCRKKLW-COOH NV1G1991 NV1D3789 333GPAPAPAQCQKWMQTCDRERKCCE GFVCTLWCRKKLW-COOH NV1G1989 NV1D3791 334GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWAAAAA-COOH NV1G1993 NV1D3792 335GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWGGGGG-COOH NV1G1967 NV1D3793 336GPCCNCSSKWCRDHSRCCGRGSAPA PAPAPAPAPAPAPAPAPGSQCQKWMQTCDRERKCCEGFVCTLWCRKKL W-COOH NV1G1969 NV1D3795 337GPCCNCSSKWCRDHSRCCGSAPAPA PAPAPAPAPAPAPAPGSQCQKWMQTCDRERKCCEGFVCTLWCRKKLW- COOH NV1G1974 NV1D3796 338GPCCNCSSKWCRDHSRCCGSAPAPA PAPAPGSQCQKWMQTCDRERKCCE GFVCTLWCRKKLW-COOHNV1G1950 NV1D3797 339 GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWGSAPAPAPAPAPAPAPAPAPAPGSCCNCSSKWCRDHSRCC- COOH NV1G1948 NV1D3798 340GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWGSAPAPAPAPAPAPAPAPAPAPGSCCNCSSKWCRDHSRCCGR- COOH NV1G2057 NV1D3799 341GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWGSAPAPAPAPAPGSCCN CSSKWCRDHSRCC-COOHNV1G1954 NV1D3800 342 GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWGSAPAPAPAPAPGSCCNCSSKWCRDHSRCCGR-COOH NV1G1956 NV1D3801 343 GPSPGARAFAPAPAPAPAPQCQKWMQTCDRERKCCEGFVCTLWCRKKL W-COOH NV1G1961 NV1D3802 344GPSPGARAFAPAPAQCQKWMQTC DRERKCCEGFVCTLWCRKKLW- COOH NV1G1960 NV1D3803345 GPSPGARAFQCQKWMQTCDRERKC CEGFVCTLWCRKKLW-COOH NV1G1977 NV1D3804 346GPDGPWRKMAPAPAPAPAPQCQK WMQTCDRERKCCEGFVCTLWCRKK LW-COOH NV1G1982NV1D3805 347 GPDGPWRKMAPAPAQCQKWMQT CDRERKCCEGFVCTLWCRKKLW- COOHNV1G1984 NV1D3806 348 GPDGPWRKMQCQKWMQTCDRER KCCEGFVCTLWCRKKLW-COOHNV1G1985 NV1D3808 349 GPFGQKASSAPAPAQCQKWMQTC DRERKCCEGFVCTLWCRKKLW-COOH NV1G1983 NV1D3809 350 GPFGQKASSQCQKWMQTCDRERKC CEGFVCTLWCRKKLW-COOHNV1G1973 NV1D3810 351 GPQRFVTGHFGGLYPANGAPAPAPA PAPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH NV1G1976 NV1D3811 352 GPQRFVTGHFGGLYPANGAPAPAQCQKWMQTCDRERKCCEGFVCTLWCR KKLW-COOH NV1G1980 NV1D3812 353GPQRFVTGHFGGLYPANGQCQKW MQTCDRERKCCEGFVCTLWCRKKL W-COOH NV1G1952NV1D3813 354 GPRRRRRRRRRRRAPAPAPAPAPQC QKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH NV1G1957 NV1D3814 355 GPRRRRRRRRRRRAPAPAQCQKWMQTCDRERKCCEGFVCTLWCRKKLW- COOH NV1G1981 NV1D3815 356GPRRRRRRRRRRRQCQKWMQTCDR ERKCCEGFVCTLWCRKKLW-COOH NV1G1959 NV1D3818 357GPYGRKKRRQRRRQCQKWMQTCD RERKCCEGFVCTLWCRKKLW-COOH NV1G1986 NV1D3819 358GPAPAPAPAPAPQCQKWMQTCDRE RKCCEGFVCTLWCRKKLW-COOH NV1G1968 NV1D3822 359GPGWCGDPGATCGKLRLYCCSGFCD SYTKTCKDKSSAGGGGSAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPGGGG SQCQKWMQTCDRERKCCEGFVCTL WCRKKLW-COOH NV1G1945NV1D3823 360 GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWGGGGSAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPGGGGSG WCGDPGATCGKLRLYCCSGFCDSYT KTCKDKSSA-COOHNV1G1972 NV1D3824 361 GPGWCGDPGATCGKLRLYCCSGFCDAYTKTCKDKSSAGGGGSAPAPAPAP APAPAPAPAPAPAPAPAPAPAPGGGGSQCQKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1946 NV1D3825 362GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWGGGGSAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPGGGGSG WCGDPGATCGKLRLYCCSGFCDAYT KTCKDKSSA-COOHNV1G1970 NV1D3826 363 GPGWCGDPGATCGKLRLYCCSGFCDCYTKTCKDKSSAGGGGSAPAPAPAP APAPAPAPAPAPAPAPAPAPAPGGGGSQCQKWMQTCDRERKCCEGFVCT LWCRKKLW-COOH NV1G1949 NV1D3828 364GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWGSGGGGSAPAPAPAPAPAPAPAPAPAPGGGGSGSCCNCSSK WCRDHSRCCGR-COOH NV1G1951 NV1D3829 365GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWGSGGGGSAPAPAPAPAPAPAPAPAPAPGGGGSGSCCNCSSK WCRDHSRCC-COOH NV1G1971 NV1D3830 366GPCCNCSSKWCRDHSRCCGRGSGG GGSAPAPAPAPAPAPAPAPAPAPGGGGSGSQCQKWMQTCDRERKCCEGF VCTLWCRKKLW-COOH NV1G1975 NV1D3832 367GPCRTIGPSVCAPAPAPAPAPAPAPA PAPAPQCQKWMQTCDRERKCCEGF VCTLWCRKKLW-COOHNV1G1978 NV1D3833 368 GPCRTIGPSVCAPAPAPAPAPQCQK WMQTCDRERKCCEGFVCTLWCRKKLW-COOH NV1G1979 NV1D3834 369 GPCRTIGPSVCAPAPAQCQKWMQTCDRERKCCEGFVCTLWCRKKLW- COOH NV1G2043 NV1D3835 370GPCRTIGPSVCQCQKWMQTCDRER KCCEGFVCTLWCRKKLW-COOH NV1G1955 NV1D3838 371GPQCQKWMQTCDRERKCCEGFVCT LWCRKKLWAPAPACRTIGPSVC- COOH

In some embodiments, the isolated Protoxin-II variant inhibits humanNav1.7 activity with an IC₅₀ value of about 3×10⁻⁸ M or less.

In some embodiments, the isolated Protoxin-II variant inhibits humanNav1.7 activity with an IC₅₀ value of between about 3×10⁻⁸ M to about1×10⁻⁹ M.

Another embodiment of the invention is an isolated Protoxin-II variantcomprising the amino acid sequenceGPQCX₁X₂WX₃QX₄Cx₅X₆X₇X₈X₉CCX₁₀X₁₁FX₁₂CX₁₃LWCX₁₄KKLW (SEQ ID NO: 404),wherein

X₁ is Q, R, K, A or S;

X₂ is K, S, Q or R;

X₃ is M or F;

X₄ is T, S, R, K or Q;

X₅ is D or T;

X₆ is S, A or R;

X₇ is E, R, N, K, T or Q;

X₈ is R or K;

X₉ is K, Q, S or A;

X₁₀ is E, Q or D;

X₁₁ is G or Q;

X₁₂ is V or S;

X₁₃ is R or T; and

X₁₄ is K or R.

Exemplary Protoxin-II variants that inhibit human Nav1.7 activity withan IC₅₀ value of about 30×10⁻⁹ M or less are variants comprising theamino acid sequences of SEQ ID NOs: 56, 78, 111, 114, 117, 118, 119,122, 123, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141,142, 145, 146, 147, 149, 150, 151, 152, 153, 154, 156, 158, 159, 165,172, 173, 175, 177, 178, 183, 184, 185, 186, 189, 190, 193, 197, 199,207, 210, 211, 216, 217, 224, 266, 273, 282 or 335.

In some embodiments, the isolated Protoxin-II variant selectivelyinhibits human Nav1.7. The Protoxin-II variants of the invention may bemore selective towards Nav1.7 when compared to the recombinantProtoxin-II (SEQ ID NO: 2). In the QPatch electrophysiology assay,recombinant Protoxin-II has an IC₅₀ of about 2.2×10⁻⁹ M for Nav1.7 andan IC₅₀ of about 62×10⁻⁹ M for Nav1.6, and therefore the ratio of IC₅₀for Nav1.6 to IC₅₀ for Nav1.7 about 28 fold. “Selectivity” or“selective” or “more selective” or “selectively blocks” or “selectivelyinhibits” when used herein refers to a Protoxin-II variant that has aratio of IC₅₀ for Nav1.6 to IC₅₀ for Nav1.7 (IC₅₀(Nav1.6)/IC₅₀(Nav1.7))equal or over about 30. IC₅₀ for Nav1.6 may be assayed in a QPatchelectrophysiology assay using cell lines stably expressing Nav1.6 usingsimilar methods to those described for Nav1.7.

Residue positions in Protoxin-II that can be mutagenized to improveselectivity include residues 7 and 19, and optionally residues 1 and 11,and further optionally 12, 20, 22 and 26 (residue numbering according toSEQ ID NO: 1). Exemplary substitutions to improve selectivity are Y1Q,W7Q, S11R, S11A, E12T, M19F, V20S, R22T, and K26R. Exemplary Protoxin-IIvariants with improved selectivity are variants of SEQ ID NOs: 56, 59,65, 78, 111, 114, 117, 118, 119, 121, 122, 123, 129, 130, 133, 150, 190,217, 281, 324, 325 or 326.

Another embodiment of the invention is an isolated Protoxin-II variantcomprising the sequence GPX₁CQKWMQX₂CDX₃X₄RKCCX₅GFX₆CX₇LWCX₈KKLW (SEQ IDNO: 405); wherein

X₁ is Y, Q, A, S or R;

-   -   X₂ is T or S;    -   X₃ is S, R or A;    -   X₄ is E, T or N;

X₅ is E or Q;

X₆ is V or S;

X₇ is R or T; and

X₈ is K or R;

wherein the Protoxin-II variant inhibits human Nav1.7 activity with anIC₅₀ value of about 3×10⁻⁹ M or less, and selectively inhibits humanNav1.7.

In some embodiments, the isolated Protoxin-II variant comprises thesequence GPQCQKWMQX₁CDX₂X₃RKCCX₄GFX₅CX₆LWCX₈KKLW (SEQ ID NO: 406);wherein

X₁ is T or S;

X₂ is S, R or A;

X₃ is E, T or N;

X₄ is E or Q;

X₅ is V or S;

X₆ is R or T; and

X₇ is K or R.

Another embodiment is an isolated Protoxin-II variant comprising theamino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to the amino acid sequence of SEQ ID NO: 78(GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH); wherein

-   -   the amino acid sequence has Q at position 1, Q at position 7 and        F at position 19, when residue numbering is according to SEQ ID        NO: 1;    -   the polypeptide inhibits human Nav1.7 activity with an IC₅₀        value of about 30×10⁻⁹ M or less, wherein the IC₅₀ value is        measured using a FLIPR® Tetra membrane depolarization assay        using fluorescence resonance energy transfer (FRET) in the        presence of 25×10⁻⁶ M 3-veratroylveracevine in HEK293 cells        stably expressing human Nav1.7; and    -   the polypeptide selectively inhibits Nav1.7.

In some embodiments, the isolated Protoxin-II variant has a freeC-terminal carboxylic acid, amide, methylamide or butylamide group,which are generated via routine synthetic methods.

Another embodiment of the invention is an isolated fusion proteincomprising the Protoxin-II variant of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 35, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368 367, 370 or 371. Such second polypeptides may be wellknown leader or secretory signal sequences, or synthetic sequencesresulting for example from cloning steps, or tags such as hexahistidinetag (SEQ ID NO: 108). Such second polypeptide may be a half-lifeextending moiety. In one embodiment, the isolated fusion proteincomprises the Protoxin-II variant of the invention conjugated to ahalf-life extending moiety.

Exemplary half-life extending moieties that can be used include wellknown human serum albumin, transthyretin (TTR), a thyroxine-bindingglobulin (TGB), albumin-binding domains, or an Fc or fragments thereof.Biologically suitable polymers or copolymers may also be used, forexample ethylene glycol or polyethylene glycol (PEG) molecules, such asPEG5000 or PEG20000, dextran, polylysine, fatty acids and fatty acidesters of different chain lengths, for example laurate, myristate,stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid,tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and thelike, octane, or carbohydrates (dextran, cellulose, oligo- orpolysaccharides). These moieties may be direct fusions with theProtoxin-II variant polypeptides and may be generated by standardcloning and expression techniques. Alternatively, well known chemicalcoupling methods may be used to attach the moieties to recombinantlyproduced Protoxin-II variants of the invention.

In another embodiment, the half-life extending moiety of the fusionprotein of the invention is human serum albumin, albumin binding domain(ABD), or polyethylene glycol (PEG).

In another embodiment, the half-life extending moiety of is conjugatedto the Protoxin-II variant via a linker. Suitable linkers are well knownand include linkers having the sequence shown in SEQ ID NOs: 80 or 81.

Exemplary fusion proteins incorporating Protoxin-II variants of theinvention are those having the polypeptide sequence of SEQ ID NOs: 83,85, 87, 89, 91, 93, 95, 97, 99, 101 or 103.

Protoxin-II variants of the invention incorporating additional moietiesmay be compared for functionality by several well-known assays. Forexample, pharmacokinetic properties of Protoxin-II variants coupled toPEG may be evaluated in well known in vivo models.

Additional Protoxin-II variants and Protoxin-II variant fusion proteinsare within the scope of the invention. Additional substitutions to theProtoxin-II variants of the invention can be made as long as theresulting variant or the fusion protein retains similar characteristicswhen compared to the parent peptide. Exemplary modifications are forexample conservative substitutions that will result in Protoxin-IIvariants with similar characteristics to those of the parent molecules.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids can be divided into four families: (1) acidic (aspartate,glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); and (4) uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. Alternatively, the amino acid repertoire can be grouped as(1) acidic (aspartate, glutamate); (2) basic (lysine, argininehistidine), (3) aliphatic (glycine, alanine, valine, leucine,isoleucine, serine, threonine), with serine and threonine optionallygrouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine,tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry,2nd ed, WH Freeman and Co., 1981). Non-conservative substitutions can bemade to the Protoxin-II variants that involve substitutions of aminoacid residues between different classes of amino acids to improveproperties of the Protoxin-II variants and Protoxin-II variant fusionproteins. Whether a change in the amino acid sequence of a polypeptideor fragment thereof results in a functional homolog can be readilydetermined by assessing the ability of the modified polypeptide orfragment to produce a response in a fashion similar to the unmodifiedpolypeptide or fragment using the assays described herein. Peptides,polypeptides or proteins in which more than one replacement takes placecan readily be tested in the same manner.

Another embodiment of the invention is an isolated syntheticpolynucleotide comprising a polynucleotide encoding the Protoxin-IIvariant of the invention.

Certain exemplary synthetic polynucleotides are disclosed herein,however, other synthetic polynucleotides which, given the degeneracy ofthe genetic code or codon preferences in a given expression system,encode the Protoxin-II variants and Protoxin-II variant fusion proteinsof the invention are also within the scope of the invention. Exemplarysynthetic polynucleotides are for example polynucleotide sequences shownin SEQ ID NOs: 84, 86, 88, 90, 92, 94, 96, 98, 100, 102 and 104, whichencode the Protoxin-II variant fusion proteins of the invention. Thoseskilled in the art can readily identify the polynucleotide segments inthe fusion proteins that encode the Protoxin-II variant itself. Thesynthetic polynucleotide sequences encoding the Protoxin-II variants orfusion proteins of the invention can be operably linked to one or moreregulatory elements, such as a promoter and enhancer, that allowexpression of the nucleotide sequence in the intended host cell. Thesynthetic polynucleotide may be a cDNA.

The polynucleotides of the invention may be produced by chemicalsynthesis such as solid phase polynucleotide synthesis on an automatedpolynucleotide synthesizer. Alternatively, the polynucleotides of theinvention may be produced by other techniques such as PCR basedduplication, vector based duplication, or restriction enzyme based DNAmanipulation techniques. Techniques for producing or obtainingpolynucleotides of known sequences are well known.

The polynucleotides of the invention may also comprise at least onenon-coding sequence, such as transcribed but not translated sequences,termination signals, ribosome binding sites, mRNA stabilizing sequences,introns and polyadenylation signals. The polynucleotide sequences mayalso comprise additional sequences encoding additional amino acids.These additional polynucleotide sequences may, for example, encode amarker or well-known tag sequences such as a hexa-histidine (SEQ ID NO:108) or a HA tag which facilitate the purification of fusedpolypeptides.

Another embodiment of the invention is a vector comprising thepolynucleotide of the invention. Such vectors may be plasmid vectors,viral vectors, vectors for baculovirus expression, transposon basedvectors or any other vector suitable for introduction of thepolynucleotide of the invention into a given organism or geneticbackground by any means. For example, polynucleotides encoding theProtoxin-II variants or the Protoxin-II variant fusion proteins of theinvention are inserted into an expression vector and may be operablylinked to control sequences in the expression vector to ensure efficientexpression, such as signal sequences, promoters (e.g. naturallyassociated or heterologous promoters), enhancer elements, andtranscription termination sequences, and are chosen to be compatiblewith the host cell chosen to express the Protoxin-II variant or theProtoxin-II variant fusion protein of the invention. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the proteinsencoded by the incorporated polynucleotides.

Suitable expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors contain selection markerssuch as ampicillin-resistance, hygromycin-resistance, tetracyclineresistance, kanamycin resistance or neomycin resistance to permitdetection of those cells transformed with the desired DNA sequences.

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, suitable promoters include, but are notlimited to, lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression ina eukaryotic cell, suitable promoters include, but are not limited to,light and/or heavy chain immunoglobulin gene promoter and enhancerelements; cytomegalovirus immediate early promoter; herpes simplex virusthymidine kinase promoter; early and late SV40 promoters; promoterpresent in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters. For expression in a yeast cell, a suitable promoter is aconstitutive promoter such as an ADH1 PGK1, ENO or PYK1 promoter and thelike, or a regulatable promoter such as a GAL1 or GAL10 promoter.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generatingrecombinant constructs. The following vectors are provided by way ofexample. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS,pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala,Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene)pSVK3, pBPV, pMSG and pSVL (Pharmacia).

An exemplary vector for expression of the Protoxin-II variants orProtoxin-II variant fusion proteins is a vector havingampicillin-resistance selection marker, CMV promoter, CMV intron, signalpeptide, neomycin resistance, fl origin of replication, SV40polyadenylation signal, and cDNA encoding the Protoxin-II variant or theProtoxin-II variant fusion protein of the invention.

Another embodiment of the invention is a host cell comprising the vectorof the invention. The term “host cell” refers to a cell into which avector has been introduced. It is understood that the term host cell isintended to refer not only to the particular subject cell but also tothe progeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not be identical to the parent cell, butare still included within the scope of the term “host cell” as usedherein. Such host cells may be eukaryotic cells, prokaryotic cells,plant cells or archeal cells.

Escherichia coli, bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species, are examples of prokaryotic host cells. Othermicrobes, such as yeast, are also useful for expression. Saccharomyces(e.g., S. cerevisiae) and Pichia are examples of suitable yeast hostcells. Exemplary eukaryotic cells may be of mammalian, insect, avian orother animal origins. Mammalian eukaryotic cells include immortalizedcell lines such as hybridomas or myeloma cell lines such as SP2/0(American Type Culture Collection (ATCC), Manassas, Va., CRL-1581), NS0(European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK,ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murinecell lines. An exemplary human myeloma cell line is U266 (ATTCCRL-TIB-196). Other useful cell lines include those derived from ChineseHamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologics,Walkersville, Md.), CHO-K1 (ATCC CRL-61) or DG44.

Introduction of a polynucleotide, such as a vector, into a host cell canbe effected by methods well known to those skilled in the art. Exemplarymethods are calcium phosphate transfection, DEAE-Dextran mediatedtransfection, microinjection, cationic lipid-mediated transfection andelectroporation.

Another embodiment of the invention is a method for producing theProtoxin-II variant of the invention comprising the steps of providing ahost cell of the invention; and culturing the host cell under conditionssufficient for the expression of at least one Protoxin-II variant of theinvention.

Host cells can be cultured under any conditions suitable for maintainingor propagating a given type of host cell and sufficient for expressing apolypeptide. Culture conditions, media, and related methods sufficientfor the expression of polypeptides are well known in the art. Forexample, many mammalian cell types can be aerobically cultured at 37° C.using appropriately buffered DMEM media while bacterial, yeast and othercell types may be cultured at 37° C. under appropriate atmosphericconditions in LB media.

In the methods of the invention, the expression of the Protoxin-IIvariant can be confirmed using a variety of well-known methods. Forexample, expression of a polypeptide can be confirmed using detectionreagents, such as using SDS-PAGE or HPLC.

Another aspect of the invention is a method of modulating the activityof Nav1.7 in a biological tissue, the method comprising contacting thebiological tissue expressing Nav1.7 with a Nav1.7-modulating amount ofthe Protoxin-II variant of the invention.

Methods of Treatment

Protoxin-II variants of the invention may be utilized in any therapywhere it is desired to treat, reduce or alleviate symptoms of pain orother disorders of sensory or sympathetic neuron dysfunction.

Pain treated with the Protoxin-II variants of the invention may be anytype of pain, such as chronic pain, acute pain, neuropathic pain,nociceptive pain, visceral pain, back pain, pain associated withinflammatory conditions, post-operative pain, thermal pain or painassociated with disease and degeneration.

Pain treated with the Protoxin-II variants of the invention may beNav1.7-mediated pain.

Nav1.7-mediated pain as used herein refers to pain resulting at leastpartially from increased Nav1.7 channel activity.

The methods of the invention may be used to treat an animal patientbelonging to any classification. Examples of such animals includemammals such as humans, rodents, dogs, cats and farm animals.

The pain and/or Nav1.7-mediated pain may result from one or more causes,such as peripheral neuropathy, central neuropathy, nerve compression orentrapment syndromes such as carpal tunnel syndrome, tarsus tunnelsyndrome, ulnar nerve entrapment, compression radiculopathy, lumbarspinal stenosis, sciatic nerve compression, spinal root compression,intercostal neuralgia, compression radiculopathy and radicular lowerback pain, spinal root lesions, neuritis, autoimmune diseases, generalinflammation, chronic inflammatory conditions, arthritis, rheumaticdiseases, lupus, osteoarthritis, general gastrointestinal disorders,colitis, gastric ulceration, duodenal ulcers, inflammatory boweldisorders, irritable bowel syndrome, pain associated with diarrhea,inflammatory eye disorders, inflammatory or unstable bladder disorders,psoriasis, skin complaints with inflammatory components, sunburn,carditis, dermatitis, myositis, neuritis, collagen vascular diseases,inflammatory pain and associated hyperalgesia and allodynia, neuropathicpain and associated hyperalgesia and allodynia, multiple sclerosis,demyelinating diseases, diabetes, diabetic neuropathy pain, causalgia,pain resulting from amputation or abscess, phantom limb pain, fracturepain, bone injury, direct trauma, HIV infection, acquired immunedeficiency syndrome (“AIDS”), small pox infection, herpes infection,exposure to toxins or other foreign particles or molecules, invasivecancer, cancer, chemotherapy, radiotherapy, hormonal therapy, burns,congenital defect, dental pain, gout pain, fibromyalgias, encephalitis,chronic alcoholism, hypothyroidism, uremia and vitamin deficiencies,trigeminal neuralgia, stroke, thalamic pain syndrome, general headache,migraine, cluster headache, tension headache, mixed-vascular andnon-vascular syndromes, sympathetically maintained pain, deafferentationsyndromes, asthma, epithelial tissue damage or dysfunction, disturbancesof visceral motility at respiratory, genitourinary, gastrointestinal orvascular regions, wounds, burns, allergic skin reactions, pruritis,vasomotor or allergic rhinitis, or bronchial disorders, dysmenorrhoea,pain during labor and delivery, dyspepsia, gastroesophageal reflux,pancreatitis, and visceralgia.

Other disorders of sensory or sympathetic neuron dysfunction that may bealleviated by the Protoxin-II variants of the invention include itch,cough and asthma. In mice, global deletion of the SCN9A gene leads tocomplete insensitivity to histamine-induced itch (Gingras et al.,American Pain Society Meeting Abstract 2013 and U.S. Pat. Publ. No.2012/0185956). This finding suggests that peptide Nav1.7 blockers mayhave utility in the treatment of itch, which may arise from varioussources, such as dermatological or inflammatory disorders; orinflammatory disorders such as renal or hepatobiliary disorders,immunological disorders, medication reactions and unknown/idiopathicconditions, including dermatitis, psoriasis, eczema, insect sting orbite. Nav1.7 is also expressed in sensory nerves innervating the airways(Muroi et al., J Physiol. 2011 Dec. 1; 589(Pt 23):5663-76; Muroi et al.,Am J Physiol Regul Integr Comp Physiol. 2013 Apr. 10), suggesting thatpeptide Nav1.7 blockers may be beneficial in the treatment of coughe.g., acute or chronic cough, or cough caused by irritation fromgastroesophageal reflux disease, and inflammatory diseases of theairways such as asthma and allergy-related immune responses,bronchospasm, chronic obstructive pulmonary disease, chronic bronchitis,emphysema, and hiccups (hiccoughs, singultus). Silencing Nav1.7 in vivoin nodose ganglia of guinea pigs using shRNA nearly abolished the coughreflex induced by mechanical probing (Muroi et al., Am J Physiol RegulIntegr Comp Physiol. 2013 Apr. 10).

One aspect of the invention is a method of alleviating or treating itch,cough or asthma in a subject by administering a therapeuticallyeffective amount of the Protoxin-II variant of the invention to asubject in need thereof for a time sufficient to alleviate the itch,cough or asthma.

Another aspect of the invention is a method of alleviating or treatingNav1.7-mediated itch, Nav1.7-mediated cough or Nav1.7-mediated asthma ina subject by administering a therapeutically effective amount of theProtoxin-II variant of the invention to a subject in need thereof for atime sufficient to alleviate the itch, cough or asthma.

Nav1.7-mediated itch as used herein refers to itch resulting at leastpartially from increased Nav1.7 channel activity.

Nav1.7-mediated cough as used herein refers to cough resulting at leastpartially from increased Nav1.7 channel activity.

Nav1.7-mediated asthma as used herein refers to asthma resulting atleast partially from increased Nav1.7 channel activity.

Protoxin-II variants of the invention may be tested for their effect inreducing or alleviating pain and/or Nav1.7-mediated pain using animalmodels described herein, and models such as the rat spinal nerveligation (SNL) model of neuropathic pain, carageenan induced allodyniamodel, the Freund's complete adjuvant (CFA)-induced allodynia model, thethermal injury model, the formalin model and the Bennett Model, andother models as described in U.S. Pat. Appl. No. 2011/0124711 and U.S.Pat. No. 7,998,980. Carageenan induced allodynia and CFA-inducedallodynia are models of inflammatory pain. The Bennett model provides ananimal model for chronic pain including post-operative pain, complexregional pain syndrome, and reflex sympathetic dystrophy.

Any of the foregoing animal models may be used to evaluate the efficacyof Protoxin-II variants of the invention inhibitor in treating painand/or NAv1.7-mediated pain. The efficacy of the Protoxin-II variants ofthe invention may be compared to a no treatment or placebo control.Additionally or alternatively, efficacy may be evaluated in comparisonto one or more known pain-relieving medicaments.

The present invention provides methods of treating Nav1.7-mediated painusing the Protoxin-II variants of the invention. It has been discoveredin the pending application by the inventors (U.S. Patent Application No.61/781,276) that administration of Nav1.7 blocking peptides areefficacious in treating and/or alleviating pain in various animal modelsof pain, contrary to what was disclosed and suggested in the literature.While peptide inhibitors of Nav1.7 have been shown to be potent and/orselective towards Nav1.7 in in vitro cell culture models usingoverexpressed Nav1.7 or on isolated neurons in which the blood-nervebarrier is subverted through desheathing or hypertonic saline injection,they have so far proven non-efficacious in in vivo animal models ofpain, where the lack of efficacy has been reported to result from theinability of the peptides to pass the blood-nerve barrier. Severalpublications describe lack of efficacy of Nav1.7 blocking peptides inanimal models of pain or in isolated nerves. For example Hackel et al.,Proc Natl Acad Sci 109:E2018-27, 2012, describes the inability ofProTx-II to inhibit action potential firing in isolated nerves unlessthe perineural barrier, which provides a diffusion barrier in thismodel, is compromised. ProTx-II was found non-efficacious in rodentmodels of acute and inflammatory pain; a likely explanation stated theinability of ProTx-II to cross the blood-nerve barrier (Schmalhofer etal., Mol Pharmacol 74:1476-1484, 2008). It has been proposed that Nav1.7peptide toxin blockers have poor oral bioavailability and they aredifficult to deliver to nerve endings, implying that their use astherapeutic agents remain limited (Dib-Hajj et al., Nature RevNeuroscience 14, 49-62, 2013).

Nav1.7 is expressed in the peripheral nervous system e.g., innociceptive dorsal root ganglions (DRG), most notably in nociceptivesmall-diameter DRG neurons, in particular in peripheral terminals in theskin, with little representation in the brain. Nav1.7 distribution (e.g.sensory ending) and physiology predispose it to a major role intransmitting painful stimuli.

One embodiment of the invention is a method of treating Nav1.7-mediatedpain by administering a therapeutically effective amount of theProtoxin-II variant of the invention to a subject in need thereof for atime sufficient to treat the Nav1.7-mediated pain.

The Protoxin-II variants of the invention Nav1.7 may be utilized in anytherapy where it is desired to treat Nav1.7-mediated pain or otherdisorders of sensory or sympathetic neuron dysfunction. “Treat” or“treatment” of pain is meant to include partially or completely toprevent, stop, inhibit, reduce, or delay the perception of pain.

In some embodiments, the Nav1.7-mediated pain is chronic pain, acutepain, neuropathic pain, nociceptive pain, visceral pain, back pain,post-operative pain, thermal pain, phantom limb pain, or pain associatedwith inflammatory conditions, primary erythemalgia (PE), paraoxysmalextreme pain disorder (PEPD), osteoarthritis, rheumatoid arthritis,lumbar discectomy, pancreatitis, fibromyalgia, painful diabeticneuropathy (PDN), post-herpetic neuropathy (PHN), trigeminal neuralgia(TN), spinal cord injuries or multiple sclerosis, or pain associatedwith disease and degeneration.

Neuropathic pain includes for example painful diabetic neuropathy (PDN),post-herpetic neuropathy (PHN) or trigeminal neuralgia (TN). Othercauses of neuropathic pain include spinal cord injuries, multiplesclerosis, phantom limb pain, post-stroke pain and HIV-associated pain.Conditions such as chronic back pain, osteoarthritis and cancer may alsoresult in the generation of neuropathic-related pain and thus arepotentially suitable for treatment with the Protoxin-II variants of theinvention.

In another embodiment, the Nav1.7-mediated pain is associated withprimary erythemalgia (PE), paraoxysmal extreme pain disorder (PEPD),osteoarthritis, rheumatoid arthritis, lumbar discectomy, pancreatitis orfibromyalgia.

In the methods of the invention, the Protoxin-II variants of theinvention may be conjugated to a second polypeptide to form a fusionprotein. Such fusion proteins are for example the well-known Fc fusionsor fusions to human serum albumin to extend half-life of the peptideinhibitors. The conjugation may be a direct conjugation via a linker,such as a glycine-serine rich linker. Such linkers are well known in theart. The Protoxin-II variants of the invention incorporating additionalmoieties may be compared for their Nav1.7 blocking ability and efficacyin treatment or reducing pain using well known methods and thosedescribed herein.

Other disorders of sensory or sympathetic neuron dysfunction that can betreated with the Protoxin-II variants of the invention, includingasthma, cough, heart-burn, itch, dermatitis, bladder instability, andReynaud's disease.

Pharmaceutical Compositions

The Protoxin-II variants of the invention may be formulated in apharmaceutically acceptable vehicle or carrier. One embodiment of theinvention is a pharmaceutical composition comprising the isolatedProtoxin-II variant of the invention and a pharmaceutically acceptableexcipient.

A suitable vehicle or carrier may be water for injection, physiologicalsaline solution or artificial cerebrospinal fluid, possibly supplementedwith other materials common in compositions for parenteraladministration. Neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. These solutions are sterile andgenerally free of particulate matter, and may be sterilized byconventional, well-known sterilization techniques (e.g., filtration).The compositions may contain pharmaceutically acceptable excipients asrequired to approximate physiological conditions, such as pH adjustingand buffering agents, stabilizing, thickening, lubricating and coloringagents, etc. Suitable vehicles and their formulation and packaging aredescribed, for example, in Remington: The Science and Practice ofPharmacy (21st ed., Troy, D. ed., Lippincott Williams & Wilkins,Baltimore, Md. (2005) Chapters 40 and 41).

In the methods of the invention, the Protoxin-II variants of theinvention may be administered by peripheral administration. “Peripheraladministration” or “administered peripherally” means introducing anagent into a subject outside of the central nervous system. Peripheraladministration encompasses any route of administration other than directadministration to the spine or brain.

Peripheral administration can be local or systemic. Local administrationmay be used to concentrate the therapeutic to the site of action, suchas local administration to joints, spinal cord, surgical wounds, sitesof injury/trauma, peripheral nerve fibers, various organs (GI,urogenital, etc) or inflamed tissues. Systemic administration results indelivery of a pharmaceutical composition to essentially the entireperipheral nervous system of the subject and may also result in deliveryto the central nervous system depending on the properties of thecomposition.

Routes of peripheral administration encompass, without limitation,topical administration, intravenous or other injection, and implantedmini-pumps or other extended release devices or formulations.

Pharmaceutical compositions of the invention include formulationsinvolving the Protoxin-II variants of the invention in sustained- orcontrolled-delivery formulations. These formulations may be achievedthrough use of for example injectable microspheres, bio-erodibleparticles, microemulsions, nanoparticles, nanocapsules, macroemulsions,polymeric compounds (such as polyesters, polyamino acids, hydrogels,poly(lactic acid), polyglycolic acid or ethylene vinylacetatecopolymers), beads or liposomes, hyaluronic acid or implantable drugdelivery devices.

The Protoxin-II variants of the invention may be prepared for use forparenteral (subcutaneous, intramuscular or intravenous), intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,intra-ocular, intra-arterial, intraportal, or intralesional routes; bysustained release systems or by implantation devices, or any otheradministration, particularly in the form of liquid solutions orsuspensions; for buccal or sublingual administration such as in the formof tablets or capsules; or intranasally such as in form of powders,nasal drops or aerosols or certain agents; transdermally in a form of agel, ointment, lotion, cream or dusting powder, suspension or patchdelivery system with chemical enhancers to either modify the skinstructure or to increase the drug concentration in the transdermalpatch, or with agents that enable the application of formulationscontaining proteins and peptides onto the skin (Int. Pat. Publ. No.WO98/53847), or applications of electric fields to create transienttransport pathways such as electroporation, or to increase the mobilityof charged drugs through the skin such as iontophoresis, or applicationof ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989 and4,767,402). The composition also may be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated.

In certain embodiments, where an implantation device is used, the devicemay be implanted into any suitable tissue or organ, and delivery of thedesired molecule may be via diffusion, timed-release bolus, orcontinuous administration.

The concentration of the Protoxin-II variants of the invention or otherpeptide inhibitors of Nav1.7 in such pharmaceutical formulation can varywidely, for example from about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%,20, or between 20 to 50, up to as much as 150, 200, 300, 400, 500, 600or 70% by weight and will be selected primarily based on fluid volumes,viscosities and other factors, according to the particular mode ofadministration selected. The Protoxin-II variants of the invention canbe lyophilized for storage and reconstituted in a suitable vehicle priorto use. This technique has been shown to be effective with conventionalprotein preparations. Lyophilization and reconstitution techniques arewell known in the art.

An exemplary pharmaceutical compositions of the present invention maycomprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH4.0-5.5, and may further include sorbitol, sucrose, Tween-20 and/or asuitable substitute thereof.

The appropriate therapeutically effective dose may be determined readilyby those skilled in the art. An effective dose refers to an amount ordosage sufficient to produce a desired result, i.e. to partially orcompletely prevent, stop, inhibit, reduce, or delay the perception ofpain associated with any painful medical condition. The effective amountmay vary depending on the specific vehicle and the Protoxin-II variantsof the invention selected, and is also dependent on a variety of factorsand conditions related to the subject to be treated and the severity ofthe pain. For example, factors such as age, weight and health of thesubject to be administered with the pharmaceutical compositions of theinvention as well as dose response curves and toxicity data obtained inpreclinical animal work could be among those considered. A determineddose may, if necessary, be repeated at appropriate time intervalsselected as appropriate by a physician or other person skilled in therelevant art (e.g. nurse, veterinarian, or veterinary technician) duringthe treatment period. The determination of an effective amount or atherapeutically effective amount for a given agent is well within theability of those skilled in the art.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 ml sterile buffered water, andbetween about 1 ng to about 100 mg, about 50 ng to about 30 mg or about5 mg to about 25 mg of a Protoxin-II variant of the invention.Similarly, a pharmaceutical composition of the invention for intravenousinfusion could be made up to contain about 250 ml of sterile Ringer'ssolution, and about 1 mg to about 30 mg or about 5 mg to about 25 mg ofthe Protoxin-II variants of the invention. Actual methods for preparingparenterally administrable compositions are well known and are describedin more detail in, for example, “Remington's Pharmaceutical Science”,15th ed., Mack Publishing Company, Easton, Pa.

The present invention will now be described with reference to thefollowing specific, non-limiting examples.

Example 1 Design and Generation of Protoxin-II Variants

Protoxin-II single position limited amino acid scanning librarysubstitution was designed to assess to what degree selectivity, peptideyield, and homogeneity can be improved.

Protoxin-II variants were designed as HRV3C protease cleavable HSAfusion proteins in the following format from N- to C-terminus:6xHis-HSA-linker-HRV3C cleavable peptide-Protoxin-II variant (“6xHis”disclosed as SEQ ID NO: 108); linker being (GGGGSGGGGSGGGGSGGGGS; SEQ IDNO: 80, HSA having the sequence of SEQ ID NO: 106, HRV3C cleavablepeptide having the sequence of SEQ ID NO: 82). Each Protoxin-II variant,after cleavage from HSA had a residual N-terminal GP from the cleavagesite.

The variants were characterized in membrane depolarization assays usingFLIPR® Tetra as described in Example 3 FLIPR® Tetra membranedepolarization assay, and in whole cell patch clamp experiments usingthe QPatch assay as described in Example 3.

Combinatorial libraries were designed to test for additive effects ofselect single position hits in an attempt to generate Nav1.7 antagonistswith further improved potency and selectivity profile compared to thenative peptide.

Construction of the Expression Vectors

The designed Protoxin-II variant genes were generated using syntheticgene assembly technology as described in U.S. Pat. No. 6,521,427. Theamino acid sequences of the designed peptide variants wereback-translated to DNA sequences using human high-frequency codons. TheDNA sequence of each variant gene, together with a portion of vector DNAincluding the DNA cloning sites, was synthesized as multipleoligonucleotides, some of which contained degenerate codons, andassembled into full-length DNA fragments. The assembled DNA fragmentswere amplified by PCR and PCR products were subsequently cloned as apool. Pooled PCR products were digested with the appropriate restrictionenzymes and cloned into the designed expression vector in such a manneras to fuse each toxin variant gene to the signal peptide and the fusionpartner (6xHis-HSA-linker-HRV3C cleavable peptide (“6xHis” disclosed asSEQ ID NO: 108) contained in the vector. Standard molecular biologytechniques were used to identify a positive clone for each designedvariant. The plasmid DNA from these positive clones was purified andsequence confirmed before expressing the Protoxin-II peptide variantfusion proteins using standard methods.

Protein Expression

HEK 293-F cells were maintained in 293 Freestyle™ media (Invitrogen Cat#12338) and split when the cell concentration was between 1.5 and2.0×10⁶ cells per ml. The cells were grown in suspension, shaking at 125RPM in a humidified incubator set at 37′C and 8% CO₂. HEK 293F cellswere transiently transfected using a DNA/lipid complex after they werediluted to 1.0×10⁶ cells per ml. To generate the complex, 1.25 μg DNAper ml of transfection was diluted in 1.0 ml of OptiPro media(Invitrogen Cat #12309) and 1.25 ml of Freestyle Max transfectionreagent (Invitrogen Cat #16447) was diluted in 1.0 ml of OptiPro media.The DNA and Max transfection reagent were mixed together and incubatedfor 10 minutes at room temperature before adding to the cells.Transfected cells were placed in a humidified incubator set at 37° C.and 8% CO₂ for 4 days shaking at 125 RPM. The supernatant was separatedfrom the cells by centrifugation at 5,000×g for 10 minutes and filteredthrough a 0.2 μm filter (Corning; Cat #431153), then concentrated 10 and50 fold using an Amicon Ultra Concentrator 10K (Cat #UFC901096), andcentrifuging for approximately 10 minutes at 3,750×g.

Example 2 Purification of Protoxin-II Variants

Protoxin-II variants were expressed as HSA fusion proteins as indicatedin Example 1 and the Protoxin-II variant peptides were cleaved withHRV3C protease prior to purification. Two methodologies were tested forefficient purification of the Protoxin-II variants.

Protein Purification Purification of Protoxin-II Variants by RP-HPLC

The secreted proteins were purified from the expression supernatants viaIMAC using 1 ml HisTrap HP columns (GE Healthcare Cat#17-5247-01). Thechromatography method was run using an AKTA Xpress and protein waseluted from the column using a step gradient of Imidazole. Peakfractions were pooled and digested overnight with HRV 3C protease (1 μgprotease/150 μg fusion).

Cleaved peptide-fusion pools were further purified using a Dionex HPLCsystem with a reverse phase Phenomenex Luna 5 μm C18(2) column(Cat#00B-4252-P0-AX). Samples were eluted from the column with a 0-68%Acetonitrile (0.05% TFA) linear gradient. Elution fractions were pooled,lyophilized overnight and reconstituted in HEPES buffered saline, pH 7.4(10 mM HEPES, 137 mM NaCl, 5.4 mM KCl, 5 mM glucose, 2 mM CaCl₂, 1 mMMgCl₂).

Table 4 shows yields of Protoxin-II variants purified by RP-HPLC. Theaverage mg yield/L was 0.01615.

TABLE 4 Protoxin-II Variant Peptide ID yield (mg) NV1D816 0.0008NV1D2511 0.0009 NV1D2513 0.0034 NV1D2504 0.0071 NV1D2260 0.0129 NV1D24980.0079 NV1D2499 0.0076 NV1D2512 0.0061 NV1D2267 0.0095 NV1D2507 0.0000NV1D2509 0.0000 NV1D2305 0.0001 NV1D815 0.0021 NV1D2506 0.0001 NV1D25050.0006 NV1D812 0.0001 NV1D2510 0.0009 NV1D769 0.0031 NV1D2497 0.0038NV1D2500 0.0004 NV1D767 0.0004 NV1D2502 0.0002 NV1D2496 0.0006 NV1D25030.0030 NV1D766 0.0054 NV1D770 0.0040 NV1D772 0.0015 NV1D792 0.0016NV1D815 0.0008 NV1D768 0.0060 NV1D2508 0.0017 NV1D2501 0.0008 NV1D22960.0018 NV1D2292 0.0059 NV1D750 0.0023 NV1D748 0.0036 NV1D774 0.0050NV1D786 0.0036 NV1D855 0.0008 NV1D2312 0.0011 NV1D1410 0.0074 NV1D14150.0128 NV1D751 0.0033

Purification of Protoxin-II Variants by Solid Phase Extraction (SPE)

The secreted proteins were purified from the expression supernatants viaIMAC using 1 ml HisTrap HP columns (GE Healthcare Cat#17-5247-01). Thechromatography method was run using an AKTA Xpress and protein waseluted from the column using a step gradient of Imidazole. Peakfractions were pooled and digested overnight with HRV3C protease (1 μgprotease/150 μg fusion). The cleaved sample was loaded into a 50 kDamolecular weight cut off centrifugal filter unit (Millipore UFC805096)and cleaved peptide collected in the filtrate fraction.

Peptide pools were loaded onto a 96-well solid phase extraction block(Agilent Bond Elut Plexa A3969030) for further purification, desalting,and concentration. Blocks were used in conjunction with a vacuummanifold (Whatman). Peptide samples were loaded and washed in 0.05% TFAin water and eluted with a step gradient of acetonitrile with 0.05% TFAin water. Elution fractions were then lyophilized overnight andreconstituted in HEPES buffered saline, pH 7.4 (10 mM HEPES, 137 mMNaCl, 5.4 mM KCl, 5 mM glucose, 2 mM CaCl₂, 1 mM MgCl₂).

Peptides were reconstituted in supplemented HEPES buffered saline, pH7.4 (10 mM HEPES, 137 mM NaCl, 5.4 mM KCl, 5 mM glucose, 2 mM CaCl₂, 1mM MgCl₂) and absorbance was measured at 280 nm. Concentration valueswere then calculated using each sample's extinction coefficient. 2 μg ofeach peptide were loaded onto an Invitrogen NuPAGE® Novex® Bis-Tris Gel15 well gel and run in MES buffer non-reduced.

Samples were analyzed on Agilent 1100 HPLC using 4-80% acetonitrile in0.05% TFA linear gradient with a Phenomenex Luna C18(2) analyticalcolumn (Cat#00A-4041-B0). Concentrations of all peptides were normalizedand 10 μl of each were injected for a total of 1.3 μg per sample.Absorbance at 220 nm was monitored and chromatograms analyzed were usingChromeleon software.

Table 5 shows yields (mg) of Protoxin-II variants purified by SPE. Theaverage mg yield/L was 0.05353.

The benefits of the SPE purification process are ease and throughput ofpurification since samples are processed in parallel in a 96-well blockrather than serially on RP-HPLC, and improvement in yield. There was, onaverage, more than 3-fold higher yield (mg/L) for variants purified bySPE versus RP-HPLC.

TABLE 5 Protoxin-II Variant Peptide ID yield (mg) NV1D12 0.0054 NV1D26590.0234 NV1D2664 0.0060 NV1D2666 0.0225 NV1D2708 0.0721 NV1D2725 0.0144NV1D2739 0.0053 NV1D2765 0.0097 NV1D2748 0.0995 NV1D2771 0.0103 NV1D27700.0121 NV1D2778 0.0644 NV1D2782 0.0202 NV1D2756 0.0466 NV1D2759 0.0218NV1D2712 0.0558 NV1D12 0.0127 NV1D2673 0.0625 NV1D2662 0.0433 NV1D26690.2661 NV1D2665 0.0389 NV1D2731 0.2547 NV1D2767 0.0238 NV1D2730 0.2566NV1D2766 0.0198 NV1D2667 0.0050 NV1D2769 0.0142 NV1D2719 0.0675 NV1D27760.0633 NV1D2663 0.0344 NV1D2709 0.1841 NV1D2720 0.0538 NV1D12 0.0095NV1D2773 0.1921 NV1D2810 0.0086 NV1D2732 0.0262 NV1D757 0.0026 NV1D7910.0206 NV1D2310 0.0085 NV1D2308 0.0179 NV1D778 0.0094 NV1D856 0.0247NV1D2309 0.0035 NV1D846 0.0043 NV1D2889 0.0107 NV1D2887 0.0061 NV1D28610.0469 NV1D2729 0.1101 NV1D2890 0.0088 NV1D2899 0.0402 NV1D2804 0.0044NV1D2734 0.0602 NV1D2772 0.2050 NV1D2775 0.2225 NV1D2738 0.0512 NV1D27400.0373 NV1D2733 0.1913 NV1D788 0.0000 NV1D757 0.0021 NV1D791 0.0007NV1D2310 0.0011 NV1D2308 0.0014 NV1D778 0.0019 NV1D2294 0.0000 NV1D8560.0047 NV1D2309 0.0023 NV1D846 0.0020 NV1D2896 0.0504 NV1D2913 0.0203NV1D2910 0.0253 NV1D2893 0.0569 NV1D2909 0.0195 NV1D2917 0.0339 NV1D29140.0201 NV1D2922 0.0554 NV1D2902 0.0061 NV1D2889 0.0022 NV1D2887 0.0025NV1D2878 0.0272 NV1D2877 0.0129 NV1D2851 0.0029 NV1D2850 0.0026 NV1D28200.0020 NV1D2819 0.0015 NV1D2814 0.0163 NV1D2918 0.0256 NV1D2921 0.0533NV1D2905 0.0126 NV1D2906 0.0189 NV1D2881 0.0207 NV1D2882 0.0223 NV1D28690.0038 NV1D2870 0.0187 NV1D2867 0.0147 NV1D2888 0.0045 NV1D2816 0.0133NV1D2885 0.0025 NV1D2974 0.0418 NV1D2972 0.1089 NV1D2971 0.0407 NV1D29700.0557 NV1D2969 0.0799

Example 3 Characterization of Protoxin-II Variants

Select Protoxin-II variants were characterized in membranedepolarization and whole cell patch clamp assays to assess their potencyand selectivity towards Nav1.7.

FLIPR® Tetra Membrane Depolarization Assay

The ability of the generated peptides to inhibit membrane depolarizationinduced by Nav1.7 agonist veratridine (3-Veratroylveracevine; Biomol,Catalog# NA125) was measured with a FRET (fluorescence resonance energytransfer) assay on FLIPR® Tetra using DISBAC2(3) (Invitrogen, K1018) asan electron acceptor and PTS18 (Trisodium8-octadecyloxypyrene-1,3,6-trisulfonate) (Sigma) as a donor by excitingthe donor at 390-420 nm and measuring FRET at 515-575 nm.

HEK293 cells stably expressing human Nav1.7 were cultured in DMEM/F-12media (1:1), supplemented with 10% fetal bovine serum, 1%penicillin/streptomycin, 400 μg/mL geneticin and 100 μM NEAAs (allreagents from Invitrogen). 50 μL of harvested cells were plated at25,000 cells/well into poly-lysine coated 384-well black clear bottomplates. The plates were incubated at room temperature (RT) for 15 minfollowed by an overnight incubation at 37° C. All incubations were donein the dark unless otherwise stated. The next day, the wells were washed4 times with assay buffer (137 mM NaCl, 4 mM KCl, 2 mM MgCl₂, 2 mMCaCl₂, 5 mM Glucose, 10 mM HEPES, pH 7.4), and resuspended in 25 μL ofassay buffer. 2× stock (6 μM) of the PTS18 dye was prepared bysuspending the dye in 10% pluronic F127 in DMSO at 1:1 (v/v ratio). 25μL of the 2×PTS18 stock was added into the wells and the cells werestained for 30 min at RT, after which the dye was washed off with theassay buffer. Peptides were suspended at 3× their final concentration inthe assay buffer containing 10 μM DISBAC2(3) and 400 μM VABSC-1 tosuppress background fluorescence (Sigma, cat#201987). 25 μL/well of thesuspended peptides were added into each well, and incubated for 60minutes at RT. Depolarization was induced by 25 μM final concentrationof veratridine (by adding 25 μL/well of 75 μM (3×) stock solution), andthe reduction in the mean intensity of FRET dye fluorescence wasmeasured 30-100 seconds after adding the agonist. A 1.3× dilution ofeach measured peptide occurred after adding veratridine by convention,the concentration at the beginning of the FLIPR® Tetra assay isreported.

Concentration-response curves of synthetic Protoxin-II (PeptideInternational) were constructed in each experimental series and wereused as controls. Fluorescence counts for each well were converted to %response by normalizing the signal to the difference between negativecontrol (response to agonist veratridine alone) and positive control(response to veratridine in the presence of 10 μM tetracaine) values.For measurements, “spatial uniformity correction” (all fluorescencetraces are normalized to the average initial starting intensity) and“subtract bias value” (subtract the initial starting intensity from eachtrace) were turned on in FLIPR® Tetra. Each data point represented theresponse in an individual well. All individual data points were used ina non-linear least-squares fitting procedure to find the best fit to aHill function using Origin (Microcal). IC₅₀ values were extracted fromthe resultant fitted curve. The mean responses of the positive (P) andnegative (N) controls were used to calculate the % response in a well asfollows: % response=100*(N−R)/(N−P).

Assay plates were accepted if the potency of control antagonists forthat day were within ±0.5 log units of their historical mean.

QPatch Assay

HEK293 cells stably expressing human Nav1.5 (SEQ ID NO: 105), Nav1.7(SEQ ID NO: 79) or Nav1.6 (SEQ ID NO: 407) were cultured in DMEM/F-12media (1:1), supplemented with 10% fetal bovine serum, 1%penicillin/streptomycin, 400 μg/mL Geneticin and 100 μM NEAAs (allreagents from Invitrogen). Cells were maintained at 37° C. and in 5% CO2and assayed upon reaching ˜50-90% confluency. CHO cells stablyexpressing human Nav1.6 in a tetracycline-inducible manner (SEQ ID NO:407) were cultured in HAMs F12, supplemented with 10% fetal bovineserum, 1% penicillin/streptomycin, 10 μg/mL Blasticidin and 400 μg/mLZeocin. Cells were maintained at 37° C. and in 5% CO2, and assayed uponreaching ˜50-90% confluency. Nav1.6 expression was induced with 1 μg/mlof tetracycline, 24-48 h prior to an experiment.

Before testing in QPatch HT (Sophion), cells were first dissociatedusing 0.05% trypsin (5 min at 37° C.), resuspended in CHO-S-SFM media(Life Technologies) and gently triturated to break up cell clumps. Celldensity was adjusted to 1-2×10⁶/mL with the same media and cells werethe transferred to a cell “hotel” in QPatch HT and used in experimentsfor several hours. For giga-ohm seal formation and whole-cell patchclamp recording, the extracellular solution contained 137 mM NaCl, 5.4mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM glucose, and 10 mM HEPES, pH=7.4and osmolarity=315 mOsm. The intracellular solution contained 135 mMCsF, 10 mM CsCl, 5 mM EGTA, 5 mM NaCl and 10 mM HEPES, pH=7.3 andosmolarity=290 mOsm. The voltage protocol used in the assay was asfollows. From a holding potential of −75 mV (Nav1.7), −60 mV (Nav1.6),or −105 mV (Nav1.5) cells were first hyperpolarized to −120 mV for 2 secand then depolarized to 0 mV for 5 ms before returning to the holdingpotential. This protocol was repeated once every 60 sec during liquidapplications (see below). Cells were otherwise held at the holdingpotential when the above voltage protocol was not executed. Uponestablishment of the whole-cell recording configuration, a total of fiveapplications of the extracellular solution (all containing 0.1% bovineserum albumin (BSA) with or without test compound, except for the lastapplication, which contained 1 μM TTX or 10 mM lidocaine as a positivecontrol) were made on to cells being recorded. The first liquidapplication contained only the control buffer (5 μl). The voltageprotocol was executed 10 times (for a total duration of 10 min) five secafter the application. The next three liquid applications (5 μl each)contained a test compound (same compound at the same concentration forall three applications) or control buffer (for control cells only). Fiveseconds after each of these applications, the voltage protocol was againexecuted 10 times (also once per min). The last application containedpositive (composed of three 10 μl sub-applications, each separated by 2sec), five seconds after which the same voltage protocol was executedtwice to obtain the baseline current. Currents were sampled at 25 kHzand filtered at 5 kHz with an 8-pole Bessle filter. The seriesresistance compensation level was set at 80%. For each cell, the peakcurrent amplitude at 0 mV for each current trace in the first fourliquid applications was first subtracted from that of the last trace inthe presence of positive control and then normalized to that of the lasttrace in the first (control buffer) application as % inhibition. Tocontrol for current rundown, this (% inhibition) value for each cell inthe presence of a test compound was further normalized to the average %inhibition value for control (typically 5-6) cells in the sameexperiment. The mean of the last two such values in the last compoundapplication (i.e., the corrected % inhibition value for eachconcentration of a test compound) were taken as the % inhibition valuefor each cell at the particular compound concentration tested. The %inhibition values for all cells tested at each compound concentrationwere averaged and used in concentration response calculations. Allexperiments were performed at room temperature (−22° C.). Data areexpressed as mean±se. Wild type Protoxin-II was included in eachexperiment as a positive control. Data were accepted only if the potencyof Protoxin-II was within ±0.5 log units of its historical mean.

IC₅₀ values for Nav1.7 for select Protoxin-II variants obtained usingthe FLIPR® Tetra are shown in Table 6.

TABLE 6 Protoxin-II variant Protoxin-II Peptide hNav1.7 Variant SEQ IDTETRA Protein ID Peptide ID NO: IC50 (nM) NV1D12_5 NV1D12 2 4.1 ± 3.6NV1G1045 NV1D791 11 4.8 ± 0.4 NV1D1332_1 NV1D1332 12 6.7 ± 0.5NV1D1336_1 NV1D1336 14 10.5 ± 1.2  NV1D1337_1 NV1D1337 15 10.3 ± 1.0 NV1G1049 NV1D2308 16 4.5 ± 0.4 NV1G953 NV1D2670 17 22.2 ± 3.3  NV1G951NV1D2674 18 4.0 ± 0.2 NV1G963 NV1D2671 20 31.5 ± 6.4  NV1G949 NV1D267521 4.3 ± 0.3 NV1G977 NV1D2665 22 4.9 ± 0.4 NV1G957 NV1D2668 23 17.5 ±2.6  NV1G965 NV1D2672 24 4.5 ± 0.3 NV1G973 NV1D2662 25 4.0 ± 0.4 NV1G975NV1D2669 26 18.4 ± 5.7  NV1G971 NV1D2673 27 4.3 ± 0.5 NV1G995 NV1D266328 4.2 ± 0.4 NV1G961 NV1D2676 29 26.5 ± 2.9  NV1G911 NV1D2666 30 66.5 ±36.7 NV1G1133 NV1D2816 31  667 ± 93.6 NV1G905 NV1D2735 32 60.0 ± 16.2NV1G979 NV1D2731 34 20.7 ± 7.2  NV1G1097 NV1D2810 35  339 ± 5750NV1G1099 NV1D2732 36  126 ± 26.9 NV1G1011 NV1D2740 37 3.6 ± 9.9 NV1G1105NV1D2729 39 8.0 ± 0.9 NV1G1013 NV1D2733 40 7.5 ± 2.9 NV1G1095 NV1D281441  754 ± 51.3 NV1G983 NV1D2730 43 25.5 ± 4.3  NV1G1003 NV1D2734 44 13.4± 0.8  NV1G1009 NV1D2738 45 2.6 ± 0.2 NV1G1129 NV1D2867 49 >1000NV1G1121 NV1D2881 50  488 ± 72.2 NV1G1123 NV1D2882 51  857 ± 65.7NV1G899 NV1D2774 52 50.5 ± 15.2 NV1G1103 NV1D2861 54 >1000 NV1G1127NV1D2870 55  784 ± 84.8 NV1G1007 NV1D2775 56 25.4 ± 2.0  NV1G1067NV1D2893 57 75.5 ± 10.5 NV1G1005 NV1D2772 59 15.6 ± 1.8  NV1G1061NV1D2896 60 80.3 ± 7.1  NV1G1085 NV1D2877 61  441 ± 73.3 NV1G1083NV1D2878 62  680 ± 40.7 NV1G1079 NV1D2889 64 12.1 ± 1.5  NV1G1001NV1D2773 65 18.8 ± 1.5  NV1G1107 NV1D2890 66 25.8 ± 4.2  NV1G1109NV1D2899 67 33.3 ± 6.7  NV1G1117 NV1D2905 68  713 ± 87.3 NV1G1119NV1D2906 69  940 ± 86.7 NV1G1115 NV1D2921 70  586 ± 71.7 NV1G1075NV1D2922 71  204 ± 45.7 NV1G1069 NV1D2909 72 97.1 ± 10.1 NV1G1065NV1D2910 73  441 ± 41.7 NV1G1063 NV1D2913 74 79.7 ± 9.3  NV1G1073NV1D2914 75  135 ± 7.8  NV1G1071 NV1D2917 76  197 ± 48.3 NV1G1113NV1D2918 77  983 ± 98.7 NV1G1153 NV1D3034 78 10.3 ± 2.1 

Select Protoxin-II variants were tested for selectivity against humanNav1.5 using QPatch. IC₅₀ values for both Nav1.7 and Nav1.5 for selectpeptides obtained using QPatch are shown in Table 7.

TABLE 7 Protoxin- II variant Protoxin-II Peptide hNav1.7 hNav1.5 VariantSEQ ID QPatch Protein ID Peptide ID NO: IC₅₀ (nM) IC₅₀ (nM) NV1D12_5NV1D12 2 2.2 ± 1.3 >1000 NV1G899 NV1D2774 52 18.7 ± 13.6 >3000 NV1G1007NV1D2775 56 4.0 ± 8.9 >3000 NV1G1005 NV1D2772 59 6.2 ± 3.2 >3000NV1G1001 NV1D2773 65 4.3 ± 3.3 >3000 NV1G1153 NV1D3034 78 4.3 ± 4.3>1000

Example 4 Generation and Characterization of Combinatorial Protoxin-IIVariants

Combinatorial libraries were designed to test for additive effects ofselect single position hits in an attempt to generate Nav1.7 antagonistswith further improved potency and selectivity profile compared to thenative peptide using several approaches.

A limited amino acid scan was conducted at all non-cysteine Protoxin-IIpositions using A, D, Q, R, K and S for diversification. In theseexperiments, Protoxin-II was expressed and tested as monovalent Fcfusion protein as described in Example 1. From this scan, substitutionsY1Q, W7Q, S11A, were identified that improved potency and/or selectivityof the resulting variants.

A full amino acid scan (excluding cys and trp) at positions M6 and M19was also conducted. M19F substitution was identified from this scan thatimproved potency and/or selectivity of the resulting variants.

Protoxin-II/Huwentoxin-IV single position chimeras were designedbidirectionally. The purpose of this library was to obtain Protoxin-IIvariants that retained potency and selectivity profile of the wild typeProtoxin-II and would achieve beneficial refolding properties associatedwith Huwentoxin-IV. Substitutions R22T and E12N were identified fromthis scan.

Peptide NV1G1153 was further engineered by diversifying position Y1 by alimited amino acid scan using R, K, T, A, D, E, Q and S, and by chargecluster engineering, where all sets of charged residues in thethree-dimensional structure of the peptide (D10/E12, K4/E17,D10/E12/R13) were mutated.

N- and C-terminal extensions were introduced to select peptides,including NV1G1153 with the purpose of improving peptide distribution tothe site of action and of improving half-life of the peptides withoutsignificantly increasing the molecular weight of the resulting peptide.The N- and C-terminal extensions that were used are shown in Table 8 and9, respectively, and are described in Oi et. al., Neuroscience Letters434, 266-272, 2008; Whitney et. al., Nature Biotechnology 2011 29:4,352-356; Sockolosky et. al., (2012) 109:40, 16095-16100. Cellpenetrating peptides HIV Tat and polyarginine were also used. Variouslinkers were used to couple the Protoxin-II variant to the N- and/orC-terminal extensions. The linkers used are shown in Table 10.

Protoxin-II variants from each campaign were tested for their potencyand selectivity for Nav1.7 using methods described in Example 3. Theamino acid sequences of the variants that inhibited Nav1.7 with an IC₅₀value of 200 nM or less are shown in Table 3. Table 11 shows the aminoacid substitutions in select variant when compared to the wild typeProtoxin-II, and the IC₅₀ values for Nav1.7 inhibition in the FLIPRTetra assay.

TABLE 8  N-terminal extension Amino acid sequence SEQ ID NO: GPAAAAA 372GPAPAPA 373 GGGGGG 374 GPCCNCSSKWCRDHSRCC 375 GPSPGARAF 376 GPDGPWRKM377 GPFGQKASS 378 GPCRTIGPSVC 379 GPSHSNTQTLAKAPEHTG 380GPQRFVTGHFGGLYPANG 381 GPGWCGDPGATCGKLRLYCCSGFCDSYTKTCKDKSSA 382APAPAPAPAP 383 GPYGRKKRRQRRR 384 GPRRRRRRRRRRR 385

TABLE 9  C-terminal extensions SEQ ID Amino acid sequence NO: CRTIGPSVC386 YGRKKRRQRRR 387 GGGGG 374 DGPWRKM 388 CCNCSSKWCRDHSRCC 389RRRRRRRRRRR 390 SHSNTQTLAKAPEHTG 391 APAPA 392 AAAAA 393 FGQKASS 394QRFVTGHFGGLYPANG 395 SPGARAF 396 GPGWCGDPGATCGKLRLYCCSGFCDAYTKTCKDKSSA397

TABLE 10  Linkers Amino acid sequence SEQ ID NO: GSAPAPAPAPAPGS 398GSAPAPAPAPAPAPAPAPAPAPGS 399 GGGGSAPAPAPAPAPAPAPAPAPAPAPAPAPA 400PAPGGGGS APAPA 392 GSGGGGSAPAPAPAPAPAPAPAPAPAPGGGGS 401 GS APAPAPAPAP383 APAPAPAPAPAPAPAPAPAP 402

TABLE 11 Protoxin-II variant Protein Nav1. Protein peptide SEQ ID 7 IC₅₀name name NO: Substitutions (nM) SE NV1G1728 NV1D3541 281 Y1A, W7Q,S11R, E12N, 9.4 1.2 M19F, R22T, K26R NV1G1870 NV1D3583 321 Y1A, W7Q,S11A, E12R, M19F, 13.1 1.57 V20S NV1G1752 NV1D3532 272 Y1A, W7Q, S11A,E12K, M19F, 17.3 2 R22T, K26R NV1G1749 NV1D3587 326 Y1A, W7Q, S11A,E12N, 18.3 2.6 M19F, V20S NV1G1725 NV1D3572 310 Y1A, W7Q, S11A, E12R,M19F, 19.8 2.2 R22T NV1G1745 NV1D3537 277 Y1A, W7Q, S11A, E12K, M19F,21.4 4.1 V20S, R22T, K26R NV1G1720 NV1D3565 304 Y1A, W7Q, S11A, E12R,M19F, 23 2.8 V20S, R22T NV1G1761 NV1D3550 290 Y1A, W7Q, S11R, M19F,R22T, 25.8 2.7 K26R NV1G1746 NV1D3576 314 Y1A, W7Q, S11A, E12N, M19F,26.7 5.2 R22T NV1G979 NV1D2731 34 Y1A, W7Q, S11A 20.7 7.2 NV1G953NV1D2670 17 Y1A, W7Q 22.2 3.3 NV1G1519 NV1D3006 133 Y1Q, W7Q, S11A,E12R, 4.03 1.05 M19F NV1G1007- NV1D2775- 111 Y1Q, W7Q, S11A, M19F 5.060.473 NH2 NH2 NV1G1517 NV1D3004 131 Y1Q, W7Q, S11R, M19F 6.23 1.56 (-GP)N-Ac- (-GP) N-Ac- 114 Y1Q, W7Q, S11A, M19F, V20S, 6.43 1.06 NV1G1137-NV1D2974- R22T NH2 NH2 NV1G1776 NV1D3339 172 Y1Q, 6.57 0.675 Q3R, W7Q,S11R, M19F, R22T, K26R NV1G1153- NV1D3034- 119 Y1Q, W7Q, S11R, M19F,R22T, 7.1 0.9 NH-methyl NH-methyl K26R (-GP) N-Ac- (-GP) N-Ac- 121 Y1Q,W7Q, S11R, M19F, R22T, 7.63 1.04 NV1G1153- NV1D3034- K26R NH2 NH2NV1G1523 NV1D3012 135 Y1Q, W7Q, S11R, E12N, M19F 7.74 0.904 NV1G1515NV1D3005 132 Y1Q, W7Q, S11A, E12N, M19F 7.83 1.38 NV1G1187 NV1D3015 138Y1Q, W7Q, S11R, M19F, K26R 8.86 2.28 NV1G1521 NV1D3018 141 Y1Q, W7Q,S11A, E12N, M19F, 9.79 2.91 K26R NV1G1267 NV1D3044 150 Y1Q, W7Q, S11R,E12N, M19F, 9.8 0.849 R22T, K26R NV1G1153 NV1D3034 78 Y1Q, W7Q, S11R,M19F, R22T, 10.3 2.14 K26R NV1G1836 NV1D3359 190 Y1Q, W7Q, T8S, S11R,M19F, 10.5 0.739 R22T, K26R NV1G1593 NV1D3050 153 Y1Q, W7Q, S11R, E12K,M19F 10.8 1.3 NV1G1215 NV1D3048 152 Y1Q, W7Q, S11A, E12K, M19F 11.1 1.05NV1G1868 NV1D3353 185 Y1Q, W7Q, T8R, S11R, M19F, 11.2 1.25 R22T, K26RNV1G1525 NV1D3013 136 Y1Q, W7Q, S11R, E12R, M19F 11.3 1.83 NV1G1775NV1D3340 173 Y1Q, Q3K, W7Q, S11R, M19F, 11.5 0.798 R22T, K26R NV1G1833NV1D3381 210 Y1Q, W7Q, S11RK14Q, M19F, 12.2 1.56 R22T, K26R NV1G1153-NV1D3034- 117 Y1Q, W7Q, S11R, M19F, R22T, 12.2 1 NH2 NH2 K26R NV1G1777NV1D3342 175 Y1Q, Q3A, W7Q, S11R, M19F, 12.8 2.67 R22T, K26R NV1G1259NV1D3058 158 Y1Q, W7Q, S11A, E12K, M19F, 12.9 1.29 R22T, K26R NV1G1511NV1D3032 146 Y1Q, W7Q, S11R, E12N, M19F, 13 203 K26R NV1G1527 NV1D3031145 Y1Q, W7Q, S11R, E12R, M19F, 13 1.36 R22T NV1G1265 NV1D3062 159 Y1Q,W7Q, S11R, E12K, M19F, 13.2 1.43 R22T, K26R NV1G1781 NV1D3388 217 Y1Q,W7Q, S11RE17Q, M19F, 13.5 1.14 R22T, K26R NV1G1824 NV1D3354 186 Y1Q,W7Q, T8K, S11R, M19F, 13.9 1.12 R22T, K26R NV1G1772 NV1D3352 184 Y1Q,K4S, W7Q, S11R, M19F, 14.2 2.01 R22T, K26R NV1G1509 NV1D3033 147 Y1Q,W7Q, S11R, E12R, M19F, 14.5 2.18 K26R NV1G1779 NV1D3351 183 Y1Q, K4Q,W7Q, S11R, M19F, 15.3 2.39 R22T, K26R NV1G1687 NV1D3526 266 Y1Q, W7Q,S11R, M19F, R22T, 15.4 K26R NV1G1269 NV1D3045 151 Y1Q, W7Q, S11R, E12R,M19F, 15.6 1.39 R22T, K26R NV1G1623 NV1D3056 156 Y1Q, W7Q, S11R, E12K,M19F, 16.2 2.99 R22T NV1G1859 NV1D3376 205 Y1Q, W7Q, S11R, K14R, M19F,16.3 2.53 R22T, K26R NV1G1153- NV1D3034- 118 Y1Q, W7Q, S11R, M19F, R22T,16.6 1.4 NH-butyl NH-butyl K26R NV1G1211 NV1D3036 149 Y1Q, W7Q, S11A,E12R, M19F, 17.2 1.55 R22T, K26R NV1G1885 NV1D3254 165 Y1Q, W7Q, S11A,M19F 17.5 2.45 NV1G1730 NV1D3542 282 Y1Q, W7Q, S11R, E12N, M19F, 17.72.5 V20S, R22T, K26R NV1G1263 NV1D3051 154 Y1Q, W7Q, S11A, E12K, M19F,17.9 1.78 R22T NV1G1818 NV1D3368 122 Y1Q, W7Q, S11R, E12T, 17.9 1.89M19F, R22T, K26R NV1G1153 NV1D3034 116 Y1Q, W7Q, S11R, M19F, R22T, 182.5 (synthetic) K26R NV1G1823 NV1D3367 197 Y1Q, W7Q, S11R, E12Q, M19F,18.6 2.17 R22T, K26R NV1G1820 NV1D3362 193 Y1Q, W7Q, D1OT, S11R, M19F,20.1 2.32 R22T, K26R NV1G1811 NV1D3369 199 Y1Q, W7Q, S11R, R13K, M19F,20.4 2.44 R22T, K26R NV1G1810 NV1D3358 189 Y1Q, W7Q, T8Q, S11R, M19F,20.5 2.11 R22T, K26R NV1G1818- NV1D3368- 123 Y1Q, W7Q, S11R, E12T, M19F,20.5 2.8 NH2 NH2 R22T, K26R NV1G1137 NV1D2974 129 Y1Q, W70, S11A, M19F,V20S, 21.6 1.34 (synthetic) R22T NV1G1221 NV1D3017 140 Y1Q, W7Q, S11A,E12R, M19F, 21.9 2.48 R22T NV1G1722 NV1D3533 273 Y1Q, W7Q, S11A, E12K,M19F, 22.4 3.5 V20S, R22T, K26R NV1G1767 NV1D3345 177 Y1Q, Q3S, W7Q,S11R, M19F, 22.4 2.52 R22T, K26R NV1G1769 NV1D3346 178 Y1Q, K4R, W7Q,S11R, M19F, 23.2 3.39 R22T, K26R NV1G1780 NV1D3387 216 Y1Q, W7Q, S11R,E17D, M19F, 23.7 2.85 R22T, K26R NV1G1886 NV1D3249 162 Y1Q, W7Q, S11A,M19F 24.1 11.5 NV1G1812 NV1D3382 211 Y1Q, W7Q, S11R, K14S, M19F, 24.32.14 R22T, K26R NV1G1857 NV1D3366 196 Y1Q, W7Q, D10S, S11R, M19F, 24.63.8 R22T, K26R NV1G1821 NV1D3378 207 Y1Q, W7Q, S11R, K14A, M19F, 24.82.66 R22T, K26R NV1G1993 NV1D3792 335 Y1Q, W7Q, S11R, M19F, R22T, 25.32.8 K26R NV1G1007 NV1D2775 56 Y1Q, W7Q, S11A, M19F 25.4 2 NV1G1787NV1D3396 224 Y1Q, W7Q, S11R, G18Q, M19 26.4 3.17 F, R22T, K26R NV1G1257NV1D3016 139 Y1Q, W7Q, S11A, E12N, M19F, 26.6 3.1 R22T NV1G1153 NV1D3034116 Y1Q, W7Q, S11R, M19F, R22T, 27.3 2.02 (synthetic) K26R NV1G1803NV1D3403 230 Y1Q, W7Q, S11R, M19F, R22T, 28.3 1.97 K26R, K27A (-GP)N-Ac- N-Ac- 115 Y1Q, W7Q, S11A, M19F, V20S, 28.6 2.23 NV1G1137 NV1D2974R22T NV1G1531 NV1D3019 142 Y1Q, W7Q, S11A, E12R, M19F, 28.7 4.78 K26RNV1G1513 NV1D3007 134 Y1Q, W7Q, S11A, M19F, K26R 29.6 9.17 NV1G1991NV1D3789 333 Y1Q, W7Q, S11R, M19F, R22T, 29.9 5.19 K26R NV1G1013NV1D2733 40 Y1R, W7Q, M19F 7.54 2.9 NV1G1740 NV1D3580 318 Y1R, W7Q,S11A, E12R, M19F, 8.4 1.5 V20S NV1G1757 NV1D3538 278 Y1R, W7Q, S11R,E12N, M19F, 11.6 1.4 R22T, K26R NV1G1741 NV1D3569 307 Y1R, W7Q, S11A,E12R, M19F, 11.9 0.8 R22T NV1G1715 NV1D3584 322 Y1R, W7Q, S11A, E12N,M19F, 13.9 1.4 V20S NV1G1754 NV1D3529 269 Y1R, W7Q, S11A, E12K, M19F,14.6 1.7 R22T, K26R NV1G1005 NV1D2772 59 Y1R, W7Q, S11A, M19F 15.6 1.8NV1G1733 NV1D3577 315 Y1R, W7Q, S11A, M19F, V20S 18.8 2.2 NV1G1744NV1D3534 274 Y1R, W7Q, S11A, E12K, M19F, 20.6 2.2 V20S, R22T, K26RNV1G1724 NV1D3562 301 Y1R, W7Q, S11A, E12R, M19F, 23.6 2.7 V20S, R22TNV1G1735 NV1D3566 305 Y1R, W7Q, S11A, M19F, R22T 23.7 2.5 NV1G1760NV1D3543 283 Y1R, W7Q, S11R, E12N, M19F, 23.8 1.9 V20S, R22T, K26RNV1G1759 NV1D3547 287 Y1R, W7Q, S11R, M19F, R22T, 26.5 2.1 K26R NV1G1751NV1D3558 297 Y1R, W7Q, S11A, E12N, M19F, 26.7 3.4 V20S, R22T NV1G1726NV1D3551 291 Y1R, W7Q, S11R, M19F, V20S, 29.3 3.8 R22T, K26R NV1G1105NV1D2729 39 Y1R, W7Q, S11A 8 8.85E−01 NV1G957 NV1D2668 23 Y1R, W7Q 17.52.6 (-GP) (-GP) 109 Y1S, W7Q, S11A, M19F 9.47 1.28 NV1G1001 NV1D2773(-GP) (-GP) 110 Y1S, W7Q, S11A, M19F 11.5 0.61 NV1G1001- NV1D2773-NH-methyl NH-methyl NV1G1003 NV1D2734 44 Y1S, W7Q, M19F 13.4 0.8NV1G1864 NV1D3581 319 Y1S, W7Q, S11A, E12R, M19F, 14.6 1.7 V20S NV1G1748NV1D3530 270 Y1S, W7Q, S11A, E12K, M19F, 15.6 2.2 R22T, K26R NV1G1758NV1D3548 288 Y1S, W7Q, S11R, M19F, R22T, 17.6 1.9 K26R NV1G1727 NV1D3544284 Y1S, W7Q, S11R, E12N, M19F, 17.8 2.2 V20S, R22T, K26R NV1G1719NV1D3570 308 Y1S, W7Q, S11A, E12R, M19F, 18.1 1.5 R22T NV1G1742 NV1D3535275 Y1S, W7Q, S11A, E12K, M19F, 18.7 2.8 V20S, R22T, K26R NV1G1001NV1D2773 65 Y1S, W7Q, S11A, M19F 18.8 1.5 NV1G1753 NV1D3585 323 Y1S,W7Q, S11A, E12N, M19F, 19.4 2.1 V20S NV1G1762 NV1D3539 279 Y1S, W7Q,S11R, E12N, M19F, 19.4 1.8 R22T, K26R NV1G1755 NV1D3574 312 Y1S, W7Q,S11A, E12N, M19F, 22.3 2.7 R22T NV1G1717 NV1D3563 302 Y1S, W7Q, S11A,E12R, M19F, 22.4 2.4 V20S, R22T NV1G1866 NV1D3559 298 Y1S, W7Q, S11A,E12N, M19F, 26.5 5.02 V20S, R22T NV1G1721 NV1D3552 292 Y1S, W7Q, S11R,M19F, V20S, 28.1 3.7 R22T, K26R NV1G975 NV1D2669 26 Y1S, W7Q 18.4 5.7NV1G983 NV1D2730 43 Y1S, W7Q, S11A 25.5 4.3 NV1G1750- NV1D3586- 325 W7Q,S11A, E12N, M19F, 4.23 0.33 NH2 NH2 V20S NV1G1747 NV1D3531 271 W7Q,S11A, E12K, M19F, R22T, 13 2.1 K26R NV1G1763 NV1D3540 280 W7Q, S11R,E12N, M19F, R22T, 16 1.5 K26R NV1G1739 NV1D3582 320 W7Q, S11A, E12R,M19F, 17.8 2.2 V20S NV1G1750 NV1D3586 324 W7Q, S11A, E12N, M19F, 20.52.2 V20S NV1G1718 NV1D3571 309 W7Q, S11A, E12R, M19F, 21 2.3 R22TNV1G1865 NV1D3560 299 W7Q, S11A, E12N, M19F, 27.2 3.42 V20S, R22TNV1G1766 NV1D3549 289 W7Q, S11R, M19F, R22T, 27.5 3.2 K26R NV1G961NV1D2676 29 W7Q, S11A 26.5 2.9 NV1G951 NV1D2674 18 Y1A, S11A 4.03 0.2NV1G1011 NV1D2740 37 Y1Q, S11A, M19F 3.62 9.9 NV1G977 NV1D2665 22 Y1Q,M19F 4.9 0.4 NV1G949 NV1D2675 21 Y1Q, S11A 4.33 0.3 NV1G973 NV1D2662 25Y1R, M19F 4.03 0.4 NV1G965 NV1D2672 24 Y1R, S11A 4.5 0.3 NV1G1009NV1D2738 45 Y1S, S11A, M19F 2.57 0.2 NV1G995 NV1D2663 28 Y1S, M19F 4.190.4 NV1G1107- NV1D2890- 112 Y1S, M6F, S11A, M19L 9.12 1.17 NH2 NH2NV1G971 NV1D2673 27 Y1S, S11A 4.31 0.5 NV1G1782 NV1D3383 212 Y1Q, W7Q,S11R, E17R, M19F, 30.3 4.06 R22T, K26R, NV1G1990 NV1D3788 332 Y1Q, W7Q,S11R, M19F, R22T, 30.3 4.78 K26R, (-GP) N-Ac- (-GP) N-Ac 120 Y1Q, W7Q,S11R, M19F, R22T, 30.4 2.96 NV1G1153- NV1D3034 K26R NV1G1786 NV1D3389218 Y1Q, W7Q, S11R, E17S, M19F, 30.8 4.48 R22T, K26R, NV1G1147 NV1D2969124 Y1S, W7Q, S11A, M19F, 31 6.15 V20S NV1G1764 NV1D3554 294 Y1A, W7Q,S11R, M19F, V20S, 31.4 3.3 R22T, K26R NV1G963 NV1D2671 20 Y1Q, W7Q 31.56.4 NV1G1835 NV1D3379 208 Y1Q, K4D, W7Q, S11R, M19F, 31.6 2.88 R22T,K26R NV1G1231 NV1D3035 148 Y1Q, W7Q, S11A, E12N, M19F, 32 4.9 R22T, K26RNV1G1743 NV1D3564 303 W7Q, S11A, E12R, M19F, 32.3 3.1 V20S, R22TNV1G1960 NV1D3803 345 Y1Q, W7Q, S11R, M19F, R22T, 32.3 5.33 K26RNV1G1924 NV1D3470 250 Y1Q, W7Q, S11R, M19L, R22T, 32.5 403 K26R NV1G1756NV1D3575 313 W7Q, S11A, E12N, M19F, R22T 33.2 3.9 NV1G1109 NV1D2899 67Y1S, W7Q, S11A, M19L 33.3 6.7 NV1G1818 NV1D3368 122 Y1Q, W7Q, S11R,E12T, M19F, 33.5 10.7 R22T, K26R NV1G1784 NV1D3386 215 Y1Q, W7Q, S11R,E17A, M19F, 33.6 4.71 R22T, K26R NV1G1141 NV1D2972 127 Y1Q, W7Q, S11A,M19F, V20S 34.1 6.2 NV1G1774 NV1D3347 179 Y1Q, K4T, W7Q, S11R, M19F,34.2 5.99 R22T, K26R NV1G1881 NV1D3257 167 Y1Q, W7Q, S11A, M19F 34.22.81 NV1G1915 NV1D3467 249 Y1Q, W7Q, S11R, E17G, M19F, 34.5 4 R22T, K26RNV1G1984 NV1D3806 348 Y1Q, W7Q, S11R, M19F, R22T, 35.1 4.56 K26RNV1G1716 NV1D3561 300 Y1A, W7Q, S11A, E12N, M19F, 35.6 5 V20S, R22T,NV1G1255 NV1D3014 137 Y1Q, W7Q, S11R, M19F, R22T 36.1 5.37 NV1G1959NV1D3818 357 Y1Q, W7Q, S11R, M19F, R22T, 36.3 204 K26R NV1G1825 NV1D3377206 Y1Q, W7Q, S11R, K14T, M19F, 36.4 4.83 R22T, K26R NV1G1723 NV1D3536276 W7Q, S11A, E12K, M19F, V20S, 37 5.4 R22T, K26R NV1G1732 NV1D3555 295Y1R, W7Q, S11A, M19F, V20S, 37.4 4.3 R22T, NV1G1983 NV1D3809 350 Y1Q,W7Q, S11R, M19F, R22T, 38.9 4.81 K26R NV1G1982 NV1D3805 347 Y1Q, W7Q,S11R, M19F, R22T, 41.2 5.44 K26R NV1G1785 NV1D3385 214 Y1Q, W7Q, S11R,E17T, M19F, 41.5 6.5 R22T, K26R NV1G1583 NV1D3030 144 Y1Q, W7Q, S11R,E12N, M19F, 41.9 5.15 R22T NV1G1729 NV1D3545 285 W7Q, S11R, E12N, M19F,V20S, 42.8 4.6 R22T, K26R NV1G1007 NV1D2775 56 Y1Q, W7Q, S11A, M19F 42.96.7 NV1G1734 NV1D3568 306 Q1A, W7Q, S11A, M19F, R22T 44 8.3 NV1G1683NV1D3523 263 Y1Q, W7Q, S11R, M19F, R22T, 44.7 K26R NV1G1834 NV1D3360 191Y1Q, W7Q, D10R, S11R, M19F, 45.2 3.79 R22T, K26R NV1G1795 NV1D3401 229Y1Q, W7Q, S11R, M19F, R22T, 45.5 6.58 K26R, K27R NV1G1689 NV1D3514 255Y1Q, W7Q, S11R, M19F, R22T, 46.4 K26R NV1G2043 NV1D3835 370 Y1Q, W7Q,S11R, M19F, R22T, 46.4 4.09 K26R NV1G1783 NV1D3384 213 Y1Q, W7Q, S11R,E17K, M19F, 46.8 7.39 R22T, K26R NV1G1239 NV1D3020 143 Y1Q, W7Q, S11A,M19F, R22T, 47.2 7.84 K26R NV1G1788 NV1D3399 227 Y1Q, W7Q, S11R, M19F,V20T, 47.3 6.36 R22T, K26R NV1G899 NV1D2774 52 Y1A, W7Q, S11A, M19F 50.515.2 NV1G2057 NV1D3799 341 Y1Q, W7Q, S11R, M19F, R22T, 50.6 6.33 K26RNV1G1738 NV1D3578 316 W7Q, S11A, M19F, V20S, 50.7 5.7 NV1G1713 NV1D3525265 Y1Q, W7Q, S11R, M19F, R22T, 52.3 K26R NV1G1765 NV1D3553 293 W7Q,S11R, M19F, V20S, R22T, 52.4 10 K26R NV1G1916 NV1D3465 247 Y1Q, W5F,W7Q, S11R, M19F, 52.8 10.3 R22T, K26R NV1G1977 NV1D3804 346 Y1Q, W7Q,S11R, M19F, R22T, 53.6 6.27 K26R NV1G1879 NV1D3259 168 Y1Q, W7Q, S11A,M19F 54.9 7.62 NV1G1884 NV1D3256 166 Y1Q, W7Q, S11A, M19F 55.7 10.5NV1G1986 NV1D3819 358 Y1Q, W7Q, S11R, M19F, R22T, 56 6.57 K26R NV1G1633NV1D3251 163 Y1Q, W7Q, S11A, M19F 56.1 13.9 NV1G1880 NV1D3261 170 Y1Q,W7Q, S11A, M19F 57 6.25 NV1G1985 NV1D3808 349 Y1Q, W7Q, S11R, M19F,R22T, 57 6.74 K26R NV1G1849 NV1D3400 228 Y1Q, W7Q, S11R, M19F, V20Q,57.3 9.52 R22T, K26R NV1G1883 NV1D3260 169 Y1Q, W7Q, S11A, M19F 57.66.91 NV1G1145 NV1D2970 125 Y1S, W7Q, S11A, M19F, R22T 58 18.8 NV1G1697NV1D3517 258 Y1Q, W7Q, S11R, M19F, R22T, 58.5 K26R NV1G1737 NV1D3579 317Y1A, W7Q, S11A, M19F, V20S 59.9 9.6 NV1G1978 NV1D3833 368 Y1Q, W7Q,S11R, M19F, R22T, 60.3 9.57 K26R NV1G1954 NV1D3800 342 Y1Q, W7Q, S11R,M19F, R22T, 60.9 6.43 K26R NV1G1989 NV1D3791 334 Y1Q, W7Q, S11R, M19F,R22T, 61.8 8.66 K26R NV1G1815 NV1D3380 209 Y1Q, K4E, W7Q, S11R, M19F, 6410.5 R22T, K26R NV1G1967 NV1D3793 336 Y1Q, W7Q, S11R, M19F, R22T, 64.68.19 K26R NV1G1869 NV1D3573 311 Y1R, W7Q, S11A, E12N, M19F, 64.7 50.7R22T NV1G1872 NV1D3777 330 Y1Q, W7Q, S11R, M19F, R22T, 64.9 15.3 K26RNV1G1979 NV1D3834 369 Y1Q, W7Q, S11R, M19F, R22T, 65.5 7.59 K26RNV1G1827 NV1D3365 195 Y1Q, W7Q, D10Q, S11R, M19F, 66.1 10.1 R22T, K26RNV1G1768 NV1D3341 174 Y1Q, Q3T, W7Q, S11R, M19F, 66.2 9.32 R22T, K26RNV1G911 NV1D2666 30 W7Q, M19F 66.5 36.7 NV1G1856 NV1D3397 225 Y1Q, W7Q,S11R, G18S, M19F, 66.7 7.31 R22T, K26R NV1G1973 NV1D3810 351 Y1Q, W7Q,S11R, M19F, R22T, 66.9 7.04 K26R NV1G1855 NV1D3398 226 Y1Q, W7Q, S11R,M19F, V20S, 67.3 11 R22T, K26R NV1G1961 NV1D3802 344 Y1Q, W7Q, S11R,M19F, R22T, 68 8.23 K26R NV1G1846 NV1D3431 244 Y1Q, K4E, W7Q, S11R,E17K, 68.6 13.9 M19F, R22T, K26R NV1G1771 NV1D3348 180 Y1Q, K4A, W7Q,S11R, M19F, 70.6 15.9 R22T, K26R NV1G1691 NV1D3520 261 Y1Q, W7Q, S11R,M19F, R22T, 71.4 K26R NV1G1681 NV1D3511 252 Y1Q, W7Q, S11R, M19F, R22T,71.5 K26R NV1G1968 NV1D3822 359 Y1Q, W7Q, S11R, M19F, R22T, 74.2 11.1K26R NV1G1813 NV1D3424 238 Y1Q, W7Q, D10K, S11R, E12K, 75.2 12.2 M19F,R22T, K26R NV1G1067 NV1D2893 57 Y1Q, W7Q, S11A, M19L 75.5 10.5 NV1G1867NV1D3546 286 Y1A, W7Q, S11R, E12N, M19F, 76 17.6 V20S, R22T, K26RNV1G1143 NV1D2971 126 Y1S, W7Q, S11A, M19F, V20S, 77.5 22.1 R22TNV1G1806 NV1D3409 232 Y1Q, W7Q, S11R, M19F, R22T, 79.1 11.3 K26R, K28TNV1G1061 NV1D2896 60 Y1R, W7Q, S11A, M19L 80.3 7.13 NV1G1793 NV1D3419236 Y1Q, W7Q, S11R, M19F, R22T, 80.9 11.9 K26R, W30D NV1G1613 NV1D3057157 Y1Q, W7Q, S11R, E12K, M19F, 83.4 16.6 K26R NV1G1585 NV1D3052 155Y1Q, W7Q, S11A, 84.8 28.8 E12K, M19F, K26R NV1G1707 NV1D3524 264 Y1Q,W7Q, S11R, M19F, R22T, 84.9 K26R NV1G1773 NV1D3350 182 Y1Q, K4E, W7Q,S11R, M19F, 85.6 14.4 R22T, K26R NV1G1949 NV1D3828 364 Y1Q, W7Q, S11R,M19F, R22T, 87.5 11 K26R NV1G1976 NV1D3811 352 Y1Q, W7Q, S11R, M19F,R22T, 87.7 15.7 K26R NV1G1956 NV1D3801 343 Y1Q, W7Q, S11R, M19F, R22T,88.1 11.4 K26R NV1G1975 NV1D3832 367 Y1Q, W7Q, S11R, M19F, R22T, 88.412.3 K26R NV1G1839 NV1D3774 328 Y1Q, W7Q, S11R, M19F, R22T, 88.6 19.6K26R NV1G1971 NV1D3830 366 Y1Q, W7Q, S11R, M19F, R22T, 88.6 9.88 K26RNV1G1882 NV1D3262 171 Y1Q, W7Q, S11A, M19F 89.2 8.32 NV1G1950 NV1D3797339 Y1Q, W7Q, S11R, M19F, R22T, 91.1 13.5 K26R NV1G1828 NV1D3363 194Y1Q, W7Q, D10A, S11R, M19F, 93.1 15.3 R22T, K26R NV1G1139 NV1D2973 128Y1Q, W7Q, S11A, M19F, R22T 93.9 19.5 NV1G1842 NV1D3430 243 Y1Q, K4D,W7Q, S11R, E17K, 93.9 14.1 M19F, R22T, K26R NV1G1948 NV1D3798 340 Y1Q,W7Q, S11R, M19F, R22T, 94.5 17.8 K26R NV1G1807 NV1D3408 231 Y1Q, W7Q,S11R, M19F, R22T, 94.8 17.8 K26R, K28R NV1G1137 NV1D2974 129 Y1Q, W7Q,S11A, M19F, V20S, 95.7 16.2 R22T NV1G1843 NV1D3432 245 Y1Q, K4E, W7Q,S11R, E17R, 95.9 10.4 M19F, R22T, K26R NV1G1822 NV1D3423 237 Y1Q, W7Q,D1OR, S11R, E12R, 99.5 9.45 M19F, R22T, K26R NV1G1862 NV1D3556 296 W7Q,S11A, M19F, V20S, R22T 100 18.5 NV1G1969 NV1D3795 337 Y1Q, W7Q, S11R,M19F, R22T, 100 14.5 K26R NV1G1980 NV1D3812 353 Y1Q, W7Q, S11R, M19F,R22T, 101 23.6 K26R NV1G1850 NV1D3414 235 Y1Q, W7Q, S11R, M19F, R22T,102 19.4 K26R, K28S NV1G1981 NV1D3815 356 Y1Q, W7Q, S11R, M19F, R22T,102 13.5 K26R NV1G1851 NV1D3390 219 Y1Q, W7Q, S11R, G18R, M19F, 108 15.5R22T, K26R NV1G1922 NV1D3466 248 Y1Q, W7Q, S11E, M19F, R22T, 108 922K26R NV1G1778 NV1D3349 181 Y1Q, K4D, W7Q, S11R, M19F, 109 16 R22T, K26RNV1G1972 NV1D3824 361 Y1Q, W7Q, S11R, M19F, R22T, 110 16.1 K26R NV1G1974NV1D3796 338 Y1Q, W7Q, S11R, M19F, R22T, 110 19.6 K26R NV1G1826 NV1D3357188 Y1Q, W7Q, T8E, S11R, M19F, 111 15.1 R22T, K26R NV1G1892 NV1D3439 246Y1Q, W7Q, S11R, M19F, R22T, 112 13.2 K26R, W30G NV1G1819 NV1D3375 204Y1Q, W7Q, S11R, R13S, M19F, 113 1270 R22T, K26R NV1G1805 NV1D3410 233Y1Q, W7Q, S11R, M19F, R22T, 114 21.5 K26R, K28A NV1G1831 NV1D3374 203Y1Q, W7Q, S11R, R13Q, M19F, 114 1600 R22T, K26R NV1G1693 NV1D3512 253Y1Q, W7Q, S11R, M19F, R22T, 115.6 K26R NV1G1854 NV1D3392 221 Y1Q, W7Q,S11R, G18T, M19F, 117 21.8 R22T, K26R NV1G1951 NV1D3829 365 Y1Q, W7Q,S11R, M19F, R22T, 122 13.3 K26R NV1G1860 NV1D3393 222 Y1Q, W7Q, S11R,G18A, M19F, 125 24.8 R22T, K26R NV1G1099 NV1D2732 36 Y1Q, W7Q, S11A 12626.9 NV1G1705 NV1D3513 254 Y1Q, W7Q, S11R, M19F, R22T, 131.2 K26RNV1G1848 NV1D3426 240 Y1Q, W7Q, D10K, S11R, E12K, 135 39.9 R13D, M19F,R22T, K26R NV1G1952 NV1D3813 354 Y1Q, W7Q, S11R, M19F, R22T, 139 30.1K26R NV1G1631 NV1D3252 164 Y1Q, W7Q, S11A, M19F 145 53 NV1G1817 NV1D3371201 Y1Q, W7Q, S11R, R13A, M19F, 151 33.7 R22T, K26R NV1G1789 NV1D3394223 Y1Q, W7Q, S11R, G18D, M19F, 155 41.4 R22T, K26R NV1G1852 NV1D3391220 Y1Q, W7Q, S11R, G18K, M19F, 157 23.1 R22T, K26R NV1G1709 NV1D3510251 Y1Q, W7Q, S11R, M19F, R22T, 159 K26R NV1G1840 NV1D3425 239 Y1Q, W7Q,D10R, S11R, 161 27.9 E12R, R13D, M19F, R22T, K26R NV1G1809 NV1D3413 234Y1Q, W7Q, S11R, M19F, R22T, 164 43.7 K26R, K28Q NV1G1863 NV1D3356 187Y1Q, W7Q, T8D, S11R, M19F, 167 32.2 R22T, K26R NV1G1699 NV1D3527 267Y1Q, W7Q, S11R, M19F, R22T, 169.1 K26R NV1G1844 NV1D3428 242 Y1Q, W7Q,D10K, S11R, E12K, 180 52.4 R13E, M19F, R22T, K26R NV1G1853 NV1D3370 200Y1Q, W7Q, S11R, R13T, M19F, 181 25.1 R22T, K26R NV1G1946 NV1D3825 362Y1Q, W7Q, S11R, M19F, R22T, 194 28.4 K26R

The wild-type Protoxin-II inhibits Nav1.7 with an IC₅₀ value of about 4nM in FLIPR assay as described in Example 3. Variants retainingsignificant Nav1.7 potency were characterized further. FIG. 1 shows thesequence genus of generated Protoxin-II variants that inhibit Nav1.7with an IC₅₀ value of 30 nM or less.

Select Protoxin-II variants were tested for their inhibition of Nav1.7and for their selectivity against human Nav1.6 using QPatch. IC₅₀ valuesfor both Nav1.7 and Nav1.6 for select peptides obtained using QPatch areshown in FIG. 2. These peptides inhibited Nav1.7 with an IC₅₀ of 30 nMor less, and were at least 30-fold selective over Nav1.7 when comparedto Nav1.6.

The amino acid sequences of the peptides shown in FIG. 2 are shown inFIG. 3. All these peptides had W7Q and M19F substitutions when comparedto the wild type Protoxin-II.

The protoxin-II variants were expressed and purified as described inExample 1, or synthesized by standard solid phase synthesis methods. Theyields of the recombinant or synthetic peptides were compared to theyields of the wild-type protoxin. Table 12 shows that the yields of theselect protoxin-II variants were significantly higher than that ofprotoxin-II, indicating improved folding properties of the variants. Thescale of the solid-phase synthesis was 0.5 mmol.

TABLE 12 Solid phase synthesis Yield Yield Recombinant Total from Fromexpression Peptide yield Crude Linear % active isomer Protoxin-II 52 mg2.7%  7.3% 54.0% NV1D2775 84 mg 4.5% 18.7% 89.1% NV1D3034 149 mg  8.0%21.0% 85.2% NV1D3368 83 mg 4.0% 24.0% 93.8%

Example 5 Protoxin-II Variants are Efficient in In Vivo Models of PainMaterials and Methods Animals

Male C57B1/6 mice (24-26 g), ordered from Charles River and housedindividually, were used for this study.

Behavioral Tests

Von Frey Test: Mechanical (tactile) threshold was assessed by Von FreyHairs following the Up-Down method (Dixon, 1980, Chaplan et al., 1994).7 graded stimuli (von Frey filaments: 0.03, 0.07, 0.16, 0.4, 0.6, 1, 2g; Stoelting, Wood Dale, Ill.) were used. Von Frey hairs were presentedperpendicularly against the center plantar area (between toris) on ahindpaw. Sufficient force was applied to bend the filament slightly andheld for 3 seconds. Per the Chaplan paper, a positive response can beeither 1) a sharp withdrawal or 2) immediate flinching upon removal ofthe filament. See Chaplan et al for more details. Mice were acclimatedto the wire mesh in the testing chamber for 30-60 minutes prior totesting.

Hargreaves Test:

A modified Hargreaves box was used to measure thermal paw withdrawallatency (PWL) (Hargreaves et al., 1988, Pain, 32:77-88; Dirig et al.,1997, J Neurosci. Methods, 76:183-191). This box consists of a chamberwith a raised glass floor maintained at a constant temperature (27° C.).The thermal nociceptive stimulus originates from a projection bulb lightbeam below the glass surface. The light beam is aimed at the areabetween toris (center plantar). The “start” button will turn on thelight and start the timer. Movements (such as a sudden withdrawal) ofthe stimulated paw will trigger the switch to turn off the light andstop the timer. The latency in seconds is displayed. If no movementoccurs, the bulb will be turned off after 20 seconds (cutoff) to preventtissue injury. The animals were allowed to habituate on the glasssurface for 30-60 minutes before PWL measurement. Constant amperage wasused throughout the study, which resulted in Pre-test paw withdrawallatencies between 8-12 seconds when averaged over 3 to 6 read-outs takenat least 5 minutes apart.

MPE % Calculation:

Percent maximum possible effect (MPE %)=(T₁−T₀)/(Tc−T₀)×100%. T₀:threshold on day0 (post-CFA, pre-pump); T₁: threshold on day1 post pumpimplantation; Tc: cut-off of the test (20 s for the Hargreaves test and2 g for the Von Frey test)

Hotplate Test:

Animals were placed on a 10″×10″ metal plate surrounded by 4 Plexiglaswalls (15 inches high). The plate was maintained at a temperature ofeither 50 or 55° C. The response latency (time when the animal firstflinches or licks its hind paw, jumps, or vocalizes) was measured andthe animal removed from the plate. Animals showing no response wereremoved from the plate after 40 s (50° C.) or 20 s (55° C.) to preventany possible tissue damage. This trial was repeated 2-5 times every15-60 minutes in a day.

Inflammatory Pain Models

CFA Model:

Animals were anesthetized with isoflurane (4% induction and 2%maintenance) and 20 μL of 100% Complete Freund's Adjuvant (CFA;Sigma-Aldrich; Saint Louis, Mo.) was injected into the center plantararea on one hind paw using a 27 gauge needle attached to a 50 μLHamilton syringe. Carrageenan model: Animals were anesthetized withisoflurane (4% induction and 2% maintenance) and 25 μL of 2%A-carrageenan (Sigma-Aldrich; Saint Louis, Mo.) dissolved in normalsaline was injected into the center plantar area on hind paws using aninsulin syringe (BD; Franklin Lakes, N.J.).

Implantation of Mini Pumps

Alzet micro-osmotic mini pumps (Durect Corporation Model 1003D and2001D) were filled and primed per manufacturer's guide. Mice wereanesthetized with isoflurane (5% induction; 2% maintenance). Their backswere shaved, wiped down with isopropyl alcohol and povidone iodine, anda small incision was made between the scapulae. Using a pair of forcepsor hemostat, a small pocket was formed by spreading the subcutaneousconnective tissues apart. The pump was inserted into the pocket with theflow moderator pointing away from the incision. The skin incision wasthen closed using 7 mm staples and the animals were allowed to recoverin their home cages.

Data Analysis

Data are represented as mean±s.e.m. Prism (Graphpad Software Inc., LaJolla, Calif.) was used for graphing and statistical analysis. Forcomparison of threshold values over time, a two-way ANOVA followed byBonferroni's multiple comparison test was used with a significance levelof p<0.05. Hotplate and MPE % data were analyzed by one-way ANOVAfollowed by Bonferroni's multiple comparison test.

Results

Efficacy of variants NV1D3034-OH (NV1D3034-COOH), NV1D3368-OH(NV1D3368-COOH) and NV1D2775-OH (NV1D2775-COOH) was studied in the CFAmodel, a commonly used model of inflammatory pain. The injection of CFAin the hindpaw induced paw edema (not shown) and hypersensitivity tothermal stimuli (thermal hyperalgesia), as indicated by the loweredthermal latency in the injected paw on day0 (FIG. 6A). Thermalhyperalgesia was completely reversed by NV1D3034-OH at 684 and 1824μg/day, when administered by a subcutaneous osmotic mini-pump (FIGS. 4Aand 4B).

NV1D3368-OH fully reversed CFA-induced thermal hyperalgesia at 684 and1824 μg/day (FIGS. 5A and 5B). NV1D2775-OH demonstrated strong efficacyin the CFA model. Thermal latencies reached values close to the cut-offfollowing NV1D2775 administration (FIGS. 6A and 6B, 1824 μg/day),suggesting a strong analgesia effect on top of the anti-hyperalgesiaeffect. In addition, NV1D2775-OH reversed CFA-induced tactile allodynia(FIGS. 6C and 6D, 1824 μg/day). The anti-hyperalgesic effect ofNV1D2775-OH was seen as early as 4 hr post-pump implantation (FIG. 7A).The effect reached the maximum at 8 hr in both the thermal and tactiletests (FIGS. 7A and 7B), which was maintained at 24 hr. Thermal latencyand tactile threshold returned the control level by 48 h post pumpimplantation (approximately 24 h after the pumps were predicted to beempty) (FIGS. 7A and 7B).

CFA-induced thermal hyperalgesia was readily reversed by two additionalpeptides, NV1D3368-amide (NV1D3368-NH₂) and NV1D3034-N-methylamide(NV1D3034-NHMe). Thermal MPE % from the experiments is summarized inTable 13.

TABLE 13 Dose (μg/day/mouse) Vehicle Peptide (PBS) 228 684 1824NV1D3034-OH 20 ± 7 (11) 22 ± 6 (6) 48 ± 10* (8) 50 ± 6* (8)  NV1D3368-OH13 ± 7 (8)  23 ± 8 (7) 42 ± 9* (7)  47 ± 6** (8) NV1D2775-OH 15 ± 4 (20)35 ± 8 (8) 57 ± 12***  85 ± 6****  (8) (12) NV1D3368-NH₂ 15 ± 13 (6) 27± 4 (4) 46 ± 9 (4)  55 ± 15 (6)  NV1D3034-  5 ± 25 (3) 49 ± 17 (6)  NHMe*P < 0.05, **P < 0.01, ***P < 0.01 and ****P < 0.0001 vs. PBS, one-wayANOVA followed by Bonferroni's multiple comparison.

NV1D2775-OH also exhibited strong, dose-dependent efficacy in thehotplate test (FIG. 8). Latencies at 50 and 55° C. reached values nearcut-off following the administration of 1824 μg/day. At 228 μg/day,NV1D2775-OH produced a modest yet significant increase in the thermallatency, compared to the PBS control.

The efficacy of NV1D2775-OH was evaluated in another model ofinflammatory pain, the carrageenan model. Animals were implanted withNV1D2775-OH or PBS pumps. Thermal withdrawal latencies were measuredpre- and on day1 post-pump. A-carrageenan was injected into the hindpawsand thermal latencies were measured again on 2, 3 and 4 hr followingcarrageenan. NV1D2775-OH at 1824 μg/day produced significant analgesia(FIG. 9). Injection of A-carrageenan in the hindpaws inducedinflammation (not shown) and lowered thermal paw withdrawal latency inthe Hargreaves test over the 4 hr test-period (FIG. 9, PBS group).Animals pretreated with NV1D2775-OH at 1824 μg/day were fully protectedfrom carrageenan-induced hyperalgesia.

We claim:
 1. An isolated Protoxin-II variant comprising the sequenceX₁X₂X₃CX₄X₅WX₆QX₇CX₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄FX₁₅CX₁₆LWCX₁₇KKLW (SEQ ID NO:403), wherein X₁ is G, P, A or deleted; X₂ is P, A or deleted; X₃ is S,Q, A, R or Y; X₄ is Q, R, K, A or S; X₅ is K, S, Q or R; X₆ is M or F;X₇ is T, S, R, K or Q; X₈ is D or T; X₉ is S, A or R; X₁₀ is E, R, N, K,T or Q; X₁₁ is R or K; X₁₂ is K, Q, S or A; X₁₃ is E, Q or D; X₁₄ is Gor Q; X₁₅ is V or S; X₁₆ is R or T; and X₁₇ is K or R; optionally havingan N-terminal extension or a C-terminal extension, wherein thepolypeptide inhibits human Nav1.7 activity with an IC₅₀ value of about1×10⁻⁷ M or less, wherein the IC₅₀ value is measured using a FLIPR®Tetra membrane depolarization assay using fluorescence resonance energytransfer (FRET) in the presence of 25×10⁻⁶ M 3-veratroylveracevine inHEK293 cells stably expressing human Nav1.7.
 2. The Protoxin-II variantof claim 1, wherein the N-terminal extension comprises the amino acidsequence of SEQ ID NOs: 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384 or
 385. 3. The Protoxin-II variant of claim 1,wherein the C-terminal extension comprises the amino acid sequence ofSEQ ID NOs: 374, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396or
 397. 4. The Protoxin-II variant of claim 2 or 3, wherein theN-terminal and/or the C-terminal extension is conjugated to theProtoxin-II variant via a linker.
 5. The Protoxin-II variant of claim 4,wherein the linker comprises the amino acid sequence of SEQ ID NOs: 383,392, 398, 399, 400, 401 or
 402. 6. The isolated Protoxin-II variant ofclaim 1 comprising the amino acid sequence of SEQ ID NOs: 30, 40, 44,52, 56, 56, 59, 65, 78, 109, 110, 111, 114, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 162, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 177, 178, 179, 180, 182, 183,184, 185, 186, 189, 190, 193, 195, 197, 199, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 224, 226, 227, 231, 232, 243,244, 245, 247, 249, 252, 255, 258, 261, 263, 264, 265, 266, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,332, 334, 335, 336, 337, 339, 340, 341, 342, 346, 351, 358, 359, 364,366, 367, or
 368. 7. The isolated Protoxin-II variant of claim 1 thatinhibits human Nav1.7 activity with an IC₅₀ value of about 3×10⁻⁸ M orless.
 8. The isolated Protoxin-II variant of claim 7 that inhibits humanNav1.7 activity with an IC₅₀ value of between about 3×10⁻⁹ M to about1×10⁻⁹ M.
 9. The isolated Protoxin-II variant of claim 7 comprising theamino acid sequence GPQCX₁X₂WX₃QX₄CX₅X₆X₇X₈X₉CCX₁₀X₁₁FX₁₂CX₁₃LWCX₁₄KKLW(SEQ ID NO: 404), wherein X₁ is Q, R, K, A or S; X₂ is K, S, Q or R; X₃is M or F; X₄ is T, S, R, K or Q; X₅ is D or T; X₆ is S, A or R; X₇ isE, R, N, K, T or Q; X₈ is R or K; X₉ is K, Q, S or A; X₁₀ is E, Q or D;X₁₁ is G or Q; X₁₂ is V or S; X₁₃ is R or T; and X₁₄ is K or R.
 10. Theisolated Protoxin-II variant of claim 9, comprising the amino acidsequence of SEQ ID NOs: 56, 78, 111, 114, 117, 118, 119, 122, 123, 129,130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 145, 146,147, 149, 150, 151, 152, 153, 154, 156, 158, 159, 165, 172, 173, 175,177, 178, 183, 184, 185, 186, 189, 190, 193, 197, 199, 207, 210, 211,216, 217, 224, 266, 273, 282 or
 335. 11. The isolated Protoxin-IIvariant of claim 7, wherein the variant selectively inhibits humanNav1.7.
 12. The isolated Protoxin-II variant of claim 11, comprising thesequence GPX₁CQKWMQX₂CDX₃X₄RKCCX₅GFX₆CX₇LWCX₈KKLW (SEQ ID NO: 405);wherein X₁ is Y, Q, A, S or R; X₂ is T or S; X₃ is S, R or A; X₄ is E, Tor N; X₅ is E or Q; X₆ is V or S; X₇ is R or T; and X₈ is K or R. 13.The isolated Protoxin-II variant of claim 12, comprising the amino acidsequence of SEQ ID NOs: 56, 59, 65, 78, 111, 114, 117, 118, 119, 121,122, 123, 129, 130, 133, 150, 190, 217, 281, 324, 325 or
 326. 14. Theisolated Protoxin-II variant of claim 12, comprising the sequenceGPQCQKWMQX₁CDX₂X₃RKCCX₄GFX₅CX₆LWCX₈KKLW (SEQ ID NO: 406); wherein X₁ isT or S; X₂ is S, R or A; X₃ is E, T or N; X₄ is E or Q; X₅ is V or S; X₆is R or T; and X₇ is K or R.
 15. An isolated Protoxin-II variantcomprising the amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:78 (GPQCQKWMQTCDRERKCCEGFVCTLWCRKKLW-COOH), wherein a) the amino acidsequence has Q at position 1, Q at position 7 and F at position 19, whenresidue numbering is according to SEQ ID NO: 1; b) the polypeptideinhibits human Nav1.7 activity with an IC₅₀ value of about 30×10⁻⁹ M orless, wherein the IC₅₀ value is measured using a FLIPR® Tetra membranedepolarization assay using fluorescence resonance energy transfer (FRET)in the presence of 25×10⁻⁶ M 3-veratroylveracevine in HEK293 cellsstably expressing human Nav1.7; and c) the polypeptide selectivelyinhibits Nav1.7.
 16. The isolated Protoxin-II variant of claim 1, 9, 14or 15, having a free C-terminal carboxylic acid, amide, methylamide orbutylamide group.
 17. An isolated fusion protein comprising theProtoxin-II variant of claim 9, 12 or 14 conjugated to a half-lifeextending moiety.
 18. The fusion protein of claim 17, wherein thehalf-life extending moiety is human serum albumin (HSA), albumin bindingdomain (ABD), Fc or polyethylene glycol (PEG).
 19. An isolatedpolynucleotide encoding the Protoxin-II variant of claim 12 or
 15. 20. Avector comprising the isolated polynucleotide of claim
 19. 21. A hostcell comprising the vector of claim
 20. 22. A method of producing theisolated Protoxin-II variant, comprising culturing the host cell ofclaim 21 and recovering the Protoxin-II variant produced by the hostcell.
 23. A pharmaceutical composition comprising the isolatedProtoxin-II variant of claim 1, 6, 12 or 15 and a pharmaceuticallyacceptable excipient.
 24. A method of treating Nav1.7-mediated pain in asubject, comprising administering to a subject in need thereof aneffective amount of the Protoxin-II variant of claim 12 or 15 to treatthe pain.
 25. The method of claim 24, wherein pain is chronic pain,acute pain, neuropathic pain, nociceptive pain, visceral pain, backpain, post-operative pain, thermal pain, phantom limb pain, or painassociated with inflammatory conditions, primary erythemalgia (PE),paraoxysmal extreme pain disorder (PEPD), osteoarthritis, rheumatoidarthritis, lumbar discectomy, pancreatitis, fibromyalgia, painfuldiabetic neuropathy (PDN), post-herpetic neuropathy (PHN), trigeminalneuralgia (TN), spinal cord injuries or multiple sclerosis.
 26. Themethod of claim 24, wherein the Protoxin-II variant is administeredperipherally.
 27. The method of claim 24, wherein the Protoxin-IIvariant is administered locally to a joint, spinal cord, surgical wound,sites of injury or trauma, peripheral nerve fibers, urogenital organs,or inflamed tissues.
 28. The method of claim 24, wherein the subject isa human.